Compositions comprising triterpenoids

ABSTRACT

The invention relates to compositions and formulations comprising at least one triterpenoic acid and at least one neutral triterpenoid and uses thereof for treating for use in treating a condition selected from Alzheimer&#39;s disease (AD), Parkinson&#39;s Diseases (PD) and vascular dementia (VD).

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/761,759, filed Mar. 20, 2018, which is a 35 U.S.C. 371 National PhaseEntry Application from PCT/IL2016/051057, filed Sep. 22, 2016, whichclaims the benefit of U.S. Provisional Patent Application No. 62/222,976filed on Sep. 24, 2015, the disclosures of which are incorporated hereinin their entirety by reference.

FIELD OF THE INVENTION

The invention relates to compositions comprising triterpenoids, and usesthereof for treating Alzheimer's disease (AD), Parkinson's Diseases (PD)and/or vascular dementia (VD).

BACKGROUND OF THE INVENTION

Various drug entities derived from plants and plant products have beendisclosed over the years, for various therapeutic applications.

For example, Paraschos et al disclose preparation of a total masticextract without polymer (TMEWP) by polar solvent extraction of crudemastic, removal of the insoluble polymer poly-β-myrcene therefrom, andseparation of acidic and neutral fractions from TMEWP (Paraschos et al(2007) Antimicrob. Agents Chemother. 51(2):551-559).

International Patent Application Publication No. WO 2005/112967 isdirected to the anticancer activity of mastic gum.

International Patent Application Publication No. WO 2010/100650 of someof the inventors of the present invention, is directed to therapeuticuses of mastic gum fractions.

International Patent Application Publication No. WO 2010/100651 of someof the inventors of the present invention, is directed to compositionsof polymeric myrcene.

International Patent Application Publication NO. WO 2012/032523 of someof the inventors of the present invention, is directed to acidiccompositions of mastic gum.

International Patent Application Publication No. WO 2005/094837 isdirected to Use of masticadienonic acid as inhibitor of DNApolymerase-beta, used for treating cancers, tumors and neurodegenerativediseases.

Marner et al (1991) disclose identification of various triterpenoidsfrom gum mastic of P. lentiscus (Marner et al (1991) Phytochemistry, 30,3709-3712).

Giner-Larza et al (2002) disclose anti-inflammatory triterpenes frompistacia terebinthus galls (Planta Med (2002), 68, 311-315).

Neurodegenerative disorders, such as Alzheimer's disease (AD),Parkinson's Diseases (PD) and vascular dementia (VD) are adult onset,chronic, progressive and irreversible severely disabling diseases.

Vascular dementia (VD) is a subtype of dementia with a prevalence thatis second only to that of Alzheimer's disease in westernized societies.VD causes many neuropsychiatric and physical problems, and represents asignificant economic burden. Brain imaging has revealed obvious changesin the cerebral cortex and white matter, and these lesions are thoughtto be the core pathology for cognitive declines in patients withvascular dementia.

Alzheimer's disease (AD) is characterized by progressive mental andcognitive deterioration with consequent formation of amyloid plaques,neurofibrillary tangles, gliosis and neuronal loss. The disease occursin both genetic and sporadic forms whose clinical course andpathological features are quite similar.

Parkinson's disease (PD) is a chronic and progressive neurodegenerativedisease caused by a selective degeneration of dopaminergic neurons inthe substantia nigra pars compacta of the brain; 80% of the neurons dieof an unknown cause before the symptoms appear. Symptoms includeintermittent tremor in the limbs, poor balance and difficulty ininitiating movement. PD has a characteristic clinical syndrome ofbradykinesia, tremor, rigidity, and postural instability. Degenerativeparkinsonian disorders can be inherited or sporadic, but are allcharacterized by neuronal loss in selective populations of vulnerableneurons. The common denominator of all degenerative parkinsoniandisorders is loss of dopaminergic neurons of the substantia nigra thatproject to the putamen (i.e., dopaminergic nigrostriatal pathway). Fivemain separate Parkinson-plus syndromes have been identified Multiplesystem atrophy (MSA), Progressive supranuclear palsy (PSP),Parkinsonism-dementia-amyotrophic lateral sclerosis complex,Corticobasal ganglionic degeneration (CBD) and Dementia with Lewy bodies(DLB). Additional Parkinson-plus syndromes include Pick's disease andolivopontocerebellar Atrophy (OPCA).

There remains an unmet need for safe, versatile and effective compoundsand compositions, which may be obtained from plants by reproducible,highly efficient and cost-effective methods, for use in the treatment ofAlzheimer's disease (AD), Parkinson's Diseases (PD) and/or vasculardementia (VD).

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described andillustrated in conjunction with systems, tools and methods which aremeant to be exemplary and illustrative, not limiting in scope.

In some embodiments, there are provided combinations of triterpenoidcompounds, compositions comprising the same and uses thereof for thetreatment of various health related conditions, such as, such asAlzheimer's disease (AD), Parkinson's Diseases (PD) and/or vasculardementia (VD).

In some embodiments, there are provided combinations of triterpenoidcompounds, compositions comprising the same and uses thereof for thewound healing and rejuvenation of a large number of cells and tissues.In some embodiments, there is provided a composition comprising acombination of at least one triterpenoic acid and at least one neutraltriterpenoid; and a pharmaceutically acceptable carrier.

In some embodiments, there is provided a composition comprising acombination of a triterpenoic acid and a neutral triterpenoid; and apharmaceutically acceptable carrier.

In some embodiments, there is provided a composition comprising orconsisting of at least one triterpenoic acid, at least one neutraltriterpenoid and a pharmaceutically acceptable carrier.

In some embodiments, the triterpenoic acid may be selected from at leastone of masticadienonic acid (MDA), isomasticadienonic acid (IMDA),masticadienolic acid (MLA), isomasticadienolic acid (IMLA), 3-O-acetylmasticadienolic acid, 3-O-acetyl epimasticadienolic acid, 3-O-acetylisomasticadienolic acid, 3-O-acetyl epi-isomasticadienolic acid,oleanonic acid (OA) and moronic acid (MA), or any combination thereof.Each possibility is a separate embodiment. In some embodiments, thetriterpenoic acid comprises or consists of at least one ofmasticadienonic acid (MDA), isomasticadienonic acid (IMDA),masticadienolic acid (MLA), isomasticadienolic acid (IMLA), 3-O-acetylmasticadienolic acid, 3-O-acetyl epimasticadienolic acid, 3-O-acetylisomasticadienolic acid, 3-O-acetyl epi-isomasticadienolic acid,oleanonic acid (OA) and moronic acid (MA), or any combination thereof.

In some embodiments, the composition comprises or consists of at leasttwo triterpenoic acids. In some embodiments, the composition comprisesor consists of at least three triterpenoic acids. In some embodiments,the composition comprises or consists of at least four triterpenoicacids. In some embodiments, the composition comprises or consists of atleast five triterpenoic acids. In some embodiments, the compositioncomprises or consists of at least six triterpenoic acids. In someembodiments, the composition comprises or consists of at least seventriterpenoic acids. In some embodiments, the composition comprises orconsists of at least eight triterpenoic acids. In some embodiments, thecomposition comprises or consists of at least nine triterpenoic acids.In some embodiments, the composition comprises or consists of at leastten triterpenoic acids. In some embodiments, the composition comprisesor consists of not more than two triterpenoic acids. In someembodiments, the composition comprises or consists of not more thanthree triterpenoic acids. In some embodiments, the composition comprisesor consists of not more than four triterpenoic acids. In someembodiments, the composition comprises or consists of not more than fivetriterpenoic acids. In some embodiments, the composition comprises orconsists of not more than six triterpenoic acids. In some embodiments,the composition comprises or consists of not more than seventriterpenoic acids. In some embodiments, the composition comprises orconsists of not more than eight triterpenoic acids. In some embodiments,the composition comprises or consists of not more than nine triterpenoicacids. In some embodiments, the composition comprises or consists notmore than ten triterpenoic acids.

In some embodiments, the at least one triterpenoic acid(s) comprises orconsists of at least MDA, IMDA, MLA, IMLA, 3-O-acetyl masticadienolicacid, 3-O-acetyl epimasticadienolic acid, 3-O-acetyl isomasticadienolicacid, 3-O-acetyl epi-isomasticadienolic acid, OA and MA. Eachpossibility is a separate embodiment. In some embodiments, thetriterpenoic acid(s) comprises or consists of at least MDA, IMDA, MLA,IMLA, 3-O-acetyl masticadienolic acid, 3-O-acetyl isomasticadienolicacid, OA and MA. In some embodiments, the triterpenoic acid(s) comprisesor consists of at least MDA, IMDA, MLA and IMLA. In some embodiments,the triterpenoic acid comprises or consists of at least MDA and IMDA. Insome embodiments, the triterpenoic acid comprises or consists of atleast MDA. In some embodiments, the triterpenoic acid comprises at leastIMDA.

In some embodiments, the triterpenoic acid is selected from MDA, IMDA,MLA, IMLA, 3-O-acetyl masticadienolic acid, 3-O-acetylepimasticadienolic acid, 3-O-acetyl isomasticadienolic acid, 3-O-acetylepi-isomasticadienolic acid, OA and MA. Each possibility is a separateembodiment. In some embodiments, the triterpenoic acid is selected fromMDA, IMDA, MLA, IMLA, 3-O-acetyl masticadienolic acid, 3-O-acetylisomasticadienolic acid, OA and MA. In some embodiments, thetriterpenoic acid is selected from MDA, IMDA, MLA and IMLA. In someembodiments, the triterpenoic acid is selected from MDA and IMDA.

In some embodiments, the triterpenoic acid consists of MDA, IMDA, MLA,IMLA, 3-O-acetyl masticadienolic acid, 3-O-acetyl epimasticadienolicacid, 3-O-acetyl isomasticadienolic acid, 3-O-acetylepi-isomasticadienolic acid, OA and MA. Each possibility is a separateembodiment. In some embodiments, the triterpenoic acid consists of MDA,IMDA, MLA, IMLA, 3-O-acetyl masticadienolic acid, 3-O-acetylisomasticadienolic acid, OA and MA. In some embodiments, thetriterpenoic acid consists of MDA, IMDA, MLA and IMLA. In someembodiments, the triterpenoic acid consists of MDA and IMDA.

In some embodiments, the neutral triterpenoid may be selected from atleast one of (8R)-3-beta, 8-dihydroxypolypoda-13E,17E,21-triene(8-dihydroxypolypoda-13E,17E,21-triene; NF-1),(8R)-3-Oxo-8-hydroxypolypoda-13E,17E,21-triene (NF-2), Oleanonicaldehyde (NF-3), Tirucallol (NF-4), 28-hydroxylup-20(29)-en-3-one(NF-A), 28-hydroxy-beta-amyrone (NF-B), 20-hydroxydammar-24-en-3-one(NF-P), 3-beta-hydroxy-13-alpha-malabarica-14(26),17E,21-triene,20-hydroxy-lupan-3-one, 28-Nor-17-hydroxylupen-3-one,28-oxo-lupen-3-one, 28-nor-beta-amyrone, Isomasticadienonic aldehyde,Isomasticadienediol, Masticadienediol, Oleanolic aldehyde(28-oxo-beta-amyrin), 3-beta-20-dihydroxylupane, Masticadienonicaldehyde, 3-oxo-malabarica-14(26),17E,21-triene, Beta-amyrone,Beta-amyrin, Germanicol, or any combination thereof. Each possibility isa separate embodiment.

In some embodiments, the neutral triterpenoid may be selected from atleast one of (8R)-3-beta, 8-dihydroxypolypoda-13E,17E,21-triene(8-dihydroxypolypoda-13E,17E,21-triene; NF-1),(8R)-3-Oxo-8-hydroxypolypoda-13E,17E,21-triene (NF-2), Oleanonicaldehyde (NF-3), Tirucallol (NF-4), 28-hydroxylup-20(29)-en-3-one(NF-A), 28-hydroxy-beta-amyrone (NF-B),3-beta-hydroxy-13-alpha-malabarica-14(26),17E,21-triene,20-hydroxy-lupan-3-one, 28-Nor-17-hydroxylupen-3-one,28-oxo-lupen-3-one, 28-nor-beta-amyrone, Isomasticadienonic aldehyde,Isomasticadienediol, Masticadienediol, Oleanolic aldehyde(28-oxo-beta-amyrin), 3-beta-20-dihydroxylupane, Masticadienonicaldehyde, 3-oxo-malabarica-14(26),17E,21-triene, Beta-amyrone,Beta-amyrin, Germanicol, or any combination thereof. Each possibility isa separate embodiment.

In some embodiments, 20-hydroxydammar-24-en-3-one (NF-P) is not presentin the pharmaceutical composition.

In some embodiments, the neutral triterpenoid comprises or consists ofat least one of (8R)-3-beta, 8-dihydroxypolypoda-13E,17E,21-triene(8-dihydroxypolypoda-13E,17E,21-triene; NF-1),(8R)-3-Oxo-8-hydroxypolypoda-13E,17E,21-triene (NF-2), Oleanonicaldehyde (NF-3), Tirucallol (NF-4), 28-hydroxylup-20(29)-en-3-one(NF-A), 28-hydroxy-beta-amyrone (NF-B), 20-hydroxydammar-24-en-3-one(NF-P), 3-beta-hydroxy-13-alpha-malabarica-14(26),17E,21-triene,20-hydroxy-lupan-3-one, 28-Nor-17-hydroxylupen-3-one,28-oxo-lupen-3-one, 28-nor-beta-amyrone, Isomasticadienonic aldehyde,Isomasticadienediol, Masticadienediol, Oleanolic aldehyde(28-oxo-beta-amyrin), 3-beta-20-dihydroxylupane, Masticadienonicaldehyde, 3-oxo-malabarica-14(26),17E,21-triene, Beta-amyrone,Beta-amyrin and Germanicol. Each possibility is a separate embodiment.

In some embodiments, the neutral triterpenoid comprises or consists ofat least one of (8R)-3-beta, 8-dihydroxypolypoda-13E,17E,21-triene(8-dihydroxypolypoda-13E,17E,21-triene; NF-1),(8R)-3-Oxo-8-hydroxypolypoda-13E,17E,21-triene (NF-2), Oleanonicaldehyde (NF-3), Tirucallol (NF-4), 28-hydroxylup-20(29)-en-3-one(NF-A), 28-hydroxy-beta-amyrone (NF-B),3-beta-hydroxy-13-alpha-malabarica-14(26),17E,21-triene,20-hydroxy-lupan-3-one, 28-Nor-17-hydroxylupen-3-one,28-oxo-lupen-3-one, 28-nor-beta-amyrone, Isomasticadienonic aldehyde,Isomasticadienediol, Masticadienediol, Oleanolic aldehyde(28-oxo-beta-amyrin), 3-beta-20-dihydroxylupane, Masticadienonicaldehyde, 3-oxo-malabarica-14(26),17E,21-triene, Beta-amyrone,Beta-amyrin and Germanicol. Each possibility is a separate embodiment.

In some embodiments, the neutral triterpenoid does not comprise20-hydroxydammar-24-en-3-one (NF-P).

In some embodiments, the neutral triterpenoid does not consist of20-hydroxydammar-24-en-3-one (NF-P).

In some embodiments the neutral triterpenoid comprises or consists of atleast two neutral triterpenoids. In some embodiments the neutraltriterpenoid comprises or consists of at least three neutraltriterpenoids. In some embodiments the neutral triterpenoid comprises orconsists of at least four neutral triterpenoids. In some embodiments theneutral triterpenoid comprises or consists of at least five neutraltriterpenoids. In some embodiments the neutral triterpenoid comprises orconsists of at least six neutral triterpenoids. In some embodiments theneutral triterpenoid comprises or consists of at least seven neutraltriterpenoids. In some embodiments the neutral triterpenoid comprises orconsists of not more than two neutral triterpenoids. In some embodimentsthe neutral triterpenoid comprises or consists of not more than threeneutral triterpenoids. In some embodiments the neutral triterpenoidcomprises or consists of not more than four neutral triterpenoids. Insome embodiments the neutral triterpenoid comprises or consists of notmore than five neutral triterpenoids. In some embodiments the neutraltriterpenoid comprises or consists of not more than six neutraltriterpenoids. In some embodiments the neutral triterpenoid comprises orconsists of not more than seven neutral triterpenoids.

In some embodiments, the neutral triterpenoid comprises at least NF-1,NF-2, NF-3, NF-4, NF-A, NF—B, NF-P,3-beta-hydroxy-13-alpha-malabarica-14(26),17E,21-triene,20-hydroxy-lupan-3-one, 28-Nor-17-hydroxylupen-3-one,28-oxo-lupen-3-one, 28-nor-beta-amyrone, Isomasticadienonic aldehyde,Isomasticadienediol, Masticadienediol, Oleanolic aldehyde(28-oxo-beta-amyrin), 3-beta-20-dihydroxylupane, Masticadienonicaldehyde, 3-oxo-malabarica-14(26),17E,21-triene, Beta-amyrone,Beta-amyrin and Germanicol. Each possibility is a separate embodiment.In some embodiments, the neutral triterpenoid comprises at least NF-1,NF-2, NF-3, NF-4, NF-A, NF—B and NF-P. In some embodiments, the neutraltriterpenoid comprises at least NF-1, NF-2, NF-3, NF-4, NF-A and NF-B.In some embodiments, the neutral triterpenoid comprises at least NF-1,NF-2, NF-3 and NF-4. In some embodiments, the neutral triterpenoidcomprises at least NF-1, NF-2 and NF-3. In some embodiments, the neutraltriterpenoid comprises at least NF-1, NF-2 and NF-4. In someembodiments, the neutral triterpenoid comprises at least NF-1, NF-3 andNF-4. In some embodiments, the neutral triterpenoid comprises at leastNF-2, NF-3 and NF-4. In some embodiments, the neutral triterpenoidcomprises at least NF-1 and NF-2. In some embodiments, the neutraltriterpenoid comprises at least NF-1. In some embodiments, the neutraltriterpenoid comprises at least NF-2. In some embodiments, the neutraltriterpenoid comprises at least NF-3. In some embodiments, the neutraltriterpenoid comprises at least NF-4. In some embodiments, the neutraltriterpenoid comprises at least NF-A. In some embodiments, the neutraltriterpenoid comprises at least NF-B.

In some embodiments, the neutral triterpenoid is selected from NF-1,NF-2, NF-3, NF-4, NF-A, NF—B, NF-P,3-beta-hydroxy-13-alpha-malabarica-14(26), 17E,21-triene,20-hydroxy-lupan-3-one, 28-Nor-17-hydroxylupen-3-one,28-oxo-lupen-3-one, 28-nor-beta-amyrone, Isomasticadienonic aldehyde,Isomasticadienediol, Masticadienediol, Oleanolic aldehyde(28-oxo-beta-amyrin), 3-beta-20-dihydroxylupane, Masticadienonicaldehyde, 3-oxo-malabarica-14(26),17E,21-triene, Beta-amyrone,Beta-amyrin and Germanicol. Each possibility is a separate embodiment.

In some embodiments, the neutral triterpenoid comprises at least NF-1,NF-2, NF-3, NF-4, NF-A, NF—B and NF-P. In some embodiments, the neutraltriterpenoid comprises at least NF-1, NF-2, NF-3, NF-4, NF-A and NF-B.In some embodiments, the neutral triterpenoid comprises at least NF-1,NF-2, NF-3 and NF-4. In some embodiments, the neutral triterpenoidcomprises at least NF-1, NF-2 and NF-3. In some embodiments, the neutraltriterpenoid comprises at least NF-1, NF-2 and NF-4. In someembodiments, the neutral triterpenoid comprises at least NF-1, NF-3 andNF-4. In some embodiments, the neutral triterpenoid comprises at leastNF-2, NF-3 and NF-4. In some embodiments, the neutral triterpenoidcomprises at least NF-1 and NF-2. In some embodiments, the neutraltriterpenoid comprises at least NF-1. In some embodiments, the neutraltriterpenoid comprises at least NF-2. In some embodiments, the neutraltriterpenoid comprises at least NF-3. In some embodiments, the neutraltriterpenoid comprises at least NF-4. In some embodiments, the neutraltriterpenoid comprises at least NF-A. In some embodiments, the neutraltriterpenoid comprises at least NF-B.

In some embodiments, the neutral triterpenoid is selected from NF-1,NF-2, NF-3, NF-4, NF-A, NF—B and NF-P. In some embodiments, the neutraltriterpenoid is selected from NF-1, NF-2, NF-3, NF-4, NF-A and NF-B. Insome embodiments, the neutral triterpenoid is selected from NF-1, NF-2,NF-3 and NF-4. In some embodiments, the neutral triterpenoid is selectedfrom NF-1, NF-2 and NF-3. In some embodiments, the neutral triterpenoidis selected from NF-1, NF-2 and NF-4. In some embodiments, the neutraltriterpenoid is selected from NF-1 and NF-2.

In some embodiments, the neutral triterpenoid consists of NF-1, NF-2,NF-3, NF-4, NF-A, NF—B, NF-P,3-beta-hydroxy-13-alpha-malabarica-14(26),17E,21-triene,20-hydroxy-lupan-3-one, 28-Nor-17-hydroxylupen-3-one,28-oxo-lupen-3-one, 28-nor-beta-amyrone, Isomasticadienonic aldehyde,Isomasticadienediol, Masticadienediol, Oleanolic aldehyde(28-oxo-beta-amyrin), 3-beta-20-dihydroxylupane, Masticadienonicaldehyde, 3-oxo-malabarica-14(26),17E,21-triene, Beta-amyrone,Beta-amyrin and Germanicol. Each possibility is a separate embodiment.In some embodiments, the neutral triterpenoid consists of NF-1, NF-2,NF-3, NF-4, NF-A, NF—B and NF-P. In some embodiments, the neutraltriterpenoid consists of NF-1, NF-2, NF-3 and NF-4. In some embodiments,the neutral triterpenoid consists of NF-1 and NF-2.

In some embodiments, there is provided a pharmaceutical compositioncomprising pharmaceutically active ingredients comprising or consistingessentially of MA, OA, MDA, IMDA 3-O-acetyl masticadienolic acid,3-O-acetyl isomasticadienolic acid, MLA, IMLA, NF-1, NF-2, NF-3 andNF-4; and a pharmaceutically acceptable carrier.

In some embodiments, there is provided a pharmaceutical compositioncomprising pharmaceutically active ingredients comprising or consistingessentially of MDA, IMDA, MLA, IMLA, NF-1, NF-2, NF-3, NF-4, NF-A andNF-B; and a pharmaceutically acceptable carrier.

In some embodiments, there is provided a pharmaceutical compositioncomprising pharmaceutically active ingredients comprising or consistingessentially of MDA, IMDA, MLA, IMLA, NF-1, NF-2, NF-3, NF-4, NF—P, NF-Aand NF-B; and a pharmaceutically acceptable carrier.

In some embodiments, there is provided a pharmaceutical compositioncomprising pharmaceutically active ingredients comprising or consistingessentially of MDA, IMDA, NF-1, NF-2, NF-3 and NF-4; and apharmaceutically acceptable carrier.

In some embodiments, there is provided a pharmaceutical compositioncomprising pharmaceutically active ingredients comprising or consistingessentially of MDA, IMDA, NF-1 and NF-2; and a pharmaceuticallyacceptable carrier.

In some embodiments, there is provided a pharmaceutical compositioncomprising pharmaceutically active ingredients comprising or consistingessentially of MDA, IMDA, NF-1, NF-2, NF-3 and NF-4; and apharmaceutically acceptable carrier.

In some embodiments, there is provided a pharmaceutical compositioncomprising pharmaceutically active ingredients comprising or consistingessentially of MA, OA, MDA, IMDA, 3-O-acetyl masticadienolic acid,3-O-acetyl isomasticadienolic acid, MLA, IMLA, NF-1, NF-2, NF-3, NF-4,NF—P, NF-A and NF-B as the sole pharmaceutically active ingredients; anda pharmaceutically acceptable carrier.

In some embodiments, there is provided a pharmaceutical compositioncomprising pharmaceutically active ingredients comprising or consistingessentially of MA, OA, MDA, IMDA, 3-O-acetyl masticadienolic acid,3-O-acetyl isomasticadienolic acid, MLA, IMLA, NF-1, NF-2, NF-3, NF-4,NF-A and NF-B as the sole pharmaceutically active ingredients; and apharmaceutically acceptable carrier.

In some embodiments, there is provided a pharmaceutical compositioncomprising pharmaceutically active ingredients comprising or consistingessentially of MDA, IMDA, NF-1, NF-2, NF-3, NF-4, NF—P, NF-A and NF-B asthe sole pharmaceutically active ingredients; and a pharmaceuticallyacceptable carrier.

In some embodiments, there is provided a pharmaceutical compositioncomprising pharmaceutically active ingredients comprising or consistingessentially of MDA, IMDA, NF-1, NF-2, NF-3, NF-4, NF-A and NF-B as thesole pharmaceutically active ingredients; and a pharmaceuticallyacceptable carrier.

In some embodiments, there is provided a pharmaceutical compositioncomprising pharmaceutically active ingredients consisting essentially ofMA, OA, MDA, IMDA, 3-O-acetyl masticadienolic acid, 3-O-acetylisomasticadienolic acid, NF-1, NF-2, NF-3, NF-4, NF-A and NF-B; and apharmaceutically acceptable carrier.

In some embodiments, there is provided a pharmaceutical compositioncomprising pharmaceutically active ingredients consisting essentially ofMA, OA, MDA, IMDA, 3-O-acetyl masticadienolic acid, 3-O-acetylisomasticadienolic acid, NF-1, NF-2, NF-3 and NF-4; and apharmaceutically acceptable carrier.

In some embodiments, there is provided a pharmaceutical compositioncomprising pharmaceutically active ingredients consisting essentially ofOA, MDA, IMDA, 3-O-acetyl masticadienolic acid, 3-O-acetylisomasticadienolic acid, NF-1, NF-2, NF-3, NF-4, NF-A and NF-B; and apharmaceutically acceptable carrier.

In some embodiments, there is provided a pharmaceutical compositioncomprising pharmaceutically active ingredients consisting essentially ofOA, MDA, IMDA, 3-O-acetyl masticadienolic acid, 3-O-acetylisomasticadienolic acid, NF-1, NF-2, NF-3 and NF-4; and apharmaceutically acceptable carrier.

In some embodiments, there is provided a pharmaceutical compositioncomprising pharmaceutically active ingredients consisting essentially ofMDA, IMDA, NF-1, NF-2; and a pharmaceutically acceptable carrier.

In some embodiments, there is provided a pharmaceutical compositioncomprising pharmaceutically active ingredients consisting essentially ofMDA, IMDA, NF-1, NF-2, NF-3, NF-4, NF-A and NF-B as the solepharmaceutically active ingredients; and a pharmaceutically acceptablecarrier.

In some embodiments, there is provided a pharmaceutical compositioncomprising pharmaceutically active ingredients consisting essentially ofMDA, IMDA, NF-1, NF-2, NF-3 and NF-4 as the sole pharmaceutically activeingredients; and a pharmaceutically acceptable carrier.

In some embodiments, there is provided a pharmaceutical compositioncomprising pharmaceutically active ingredients consisting essentially ofMDA, IMDA, NF-1 and NF-2 as the sole pharmaceutically activeingredients; and a pharmaceutically acceptable carrier.

In some embodiments, there is provided a composition comprising acombination of at least one triterpenoic acid and at least one neutraltriterpenoid, and a pharmaceutically acceptable carrier, wherein thetriterpenoic acid is selected from MDA, IMDA or both, wherein theneutral triterpenoid is selected from NF-1, NF-2, or both.

In some embodiments the composition further comprises at least oneadditional triterpenoic acid. In some embodiments, the additionaltriterpenoic acid is selected from the group consisting of MLA, IMLA,3-O-acetyl masticadienolic acid, 3-O-acetyl epimasticadienolic acid,3-O-acetyl isomasticadienolic acid, 3-O-acetyl epi-isomasticadienolicacid, OA, MA and combinations thereof.

In some embodiments, the composition further comprises at least oneadditional neutral triterpenoid. In some embodiments, the additionalneutral triterpenoid is selected from the group consisting of NF-3,NF-4, NF-A, NF—B, NF—P and combinations thereof.

In some embodiments, the composition further comprises at least oneadditional neutral triterpenoid. In some embodiments, the additionalneutral triterpenoid is selected from the group consisting of NF-3,NF-4, NF-A, NF—B and combinations thereof.

In some embodiments, the composition further comprises NF-P.

In some embodiments, at least one of the additional neutraltriterpenoids is selected from NF-3 and NF-4.

In some embodiments, the composition comprises at least two additionalneutral triterpenoids.

In some embodiments, the triterpenoic acid(s) may be obtained from aplant source. In some embodiments, any one of the triterpenoic acids maybe obtained from a plant source. In some embodiments, at least onetriterpenoic acid may be obtained from a plant source. In someembodiments, the neutral triterpenoid(s) may be obtained from a plantsource. In some embodiments, any one of the neutral triterpenoids may beobtained from a plant source. In some embodiments, at least one neutraltriterpenoid may be obtained from a plant source. In some embodiments,the plant source may include mastic gum.

In some embodiments, the triterpenoic acid(s) may be obtained via achemical synthesis. In some embodiments, any one of the triterpenoicacids may be obtained via a chemical synthesis. In some embodiments, atleast one triterpenoic acid may be obtained via a chemical synthesis. Insome embodiments, the neutral triterpenoid(s) may be obtained via achemical synthesis. In some embodiments, any one of the neutraltriterpenoids may be obtained via a chemical synthesis. In someembodiments, at least one neutral triterpenoid may be obtained via achemical synthesis.

In some embodiments, the compositions and/or combinations of compounds,as disclosed herein, unexpectedly exhibit a variety of beneficialbiological activities, which are exploited for therapeutic applicationsin a surprisingly efficient manner. More specifically, the compositionsand combinations disclosed herein are shown to be active and useful intreating conditions such as Alzheimer's disease (AD), Parkinson'sDiseases (PD) and vascular dementia (VD). In some embodiments, thetreating of Alzheimer's disease (AD), Parkinson's Diseases (PD) andvascular dementia (VD) may be associated with reversal of the condition.In some embodiments, the treating Alzheimer's disease (AD), Parkinson'sDiseases (PD) and vascular dementia (VD) may be associated with reducingor eliminating side effects caused by the condition.

In some embodiments, the compositions and/or combinations of compounds,as disclosed herein, unexpectedly exhibit a variety of beneficialbiological activities, which are exploited for therapeutic applicationsin a surprisingly efficient manner. More specifically, the compositionsand combinations disclosed herein are shown to be active and useful intreating conditions such as Multi System Athrophy (MSA) and ProgressiveSupranuclear Palsy. In some embodiments, the treating of Multi SystemAthrophy (MSA) and Progressive Supranuclear Palsy may be associated withreversal of the condition. In some embodiments, the treating of MultiSystem Athrophy (MSA) and Progressive Supranuclear Palsy may beassociated with reducing or eliminating side effects caused by thecondition.

In some embodiments, the compositions and/or combinations of compounds,as disclosed herein, unexpectedly exhibit a variety of beneficialbiological activities, which are exploited for therapeutic applicationsin a surprisingly efficient manner. More specifically, the compositionsand combinations disclosed herein are shown to be active and useful intreating conditions such as tauopathic diseases and conditions. In someembodiments, the treating of tauopathic diseases and conditions andvascular dementia (VD) may be associated with reversal of the condition.In some embodiments, the treating tauopathic diseases and conditions maybe associated with reducing or eliminating side effects caused by thecondition.

In some embodiments, there is provided a method of treating Alzheimer'sdisease (AD), Parkinson's Diseases (PD) and/or vascular dementia (VD),comprising administering to a subject a composition as disclosed herein.In some embodiments, there is provided a method of treating Alzheimer'sdisease (AD), Parkinson's Diseases (PD) and/or vascular dementia (VD) ina subject in need thereof, comprising administering to a subject atherapeutically effective amount of a composition as disclosed herein.In some embodiments, the method is for treating Alzheimer's disease(AD). In some embodiments, the method is for treating Parkinson'sDiseases (PD). In some embodiments, the method is for treating vasculardementia (VD).

In some embodiments, there is provided a method of treating Multi SystemAtrophy (MSA) and Progressive Supranuclear Palsy (PSP), comprisingadministering to a subject a composition as disclosed herein. In someembodiments, there is provided a method of treating Multi System Atrophy(MSA) and Progressive Supranuclear Palsy (PSP), in a subject in needthereof, comprising administering to a subject a therapeuticallyeffective amount of a composition as disclosed herein. In someembodiments, the method is for treating Multi System Atrophy (MSA). Insome embodiments, the method is for treating Progressive SupranuclearPalsy (PSP).

In some embodiments, there is provided a method of treating tauopathicdiseases and conditions, comprising administering to a subject acomposition as disclosed herein. In some embodiments, there is provideda method of treating tauopathic diseases and conditions, in a subject inneed thereof, comprising administering to a subject a therapeuticallyeffective amount of a composition as disclosed herein.

In some embodiments, there is provided a method of treating primaryage-related tauopathy (PART), comprising administering to a subject acomposition as disclosed herein. In some embodiments, there is provideda method of treating primary age-related tauopathy (PART), in a subjectin need thereof, comprising administering to a subject a therapeuticallyeffective amount of a composition as disclosed herein.

In some embodiments, the composition is used for inducing or promotinglife span extension in animals. In some embodiments, the animals areselected from the group of humans, non-human mammals, birds and fish.

In some embodiments, there is provided a kit comprising a pharmaceuticalcomposition as disclosed herein.

In some embodiments, there is provided a kit comprising: (a) apharmaceutical composition comprising at least one triterpenoic acid anda pharmaceutically acceptable carrier; (b) a pharmaceutical compositioncomprising at least one neutral triterpenoid and a pharmaceuticallyacceptable carrier.

In some embodiments, there is provided a use of at least onetriterpenoic acid and at least one neutral triterpenoid in thepreparation of a composition for treating Alzheimer's disease (AD),Parkinson's Diseases (PD) and/or vascular dementia (VD).

In some embodiments, there is provided a use of at least onetriterpenoic acid and at least one neutral triterpenoid in thepreparation of a composition for treating Multi System Atrophy (MSA) andProgressive Supranuclear Palsy (PSP).

In some embodiments, there is provided a use of at least onetriterpenoic acid and at least one neutral triterpenoid in thepreparation of a composition for treating tauopathic diseases andconditions.

In some embodiments, there is provided a use of at least onetriterpenoic acid and at least one neutral triterpenoid in thepreparation of a composition for treating tauopathic diseases andconditions.

In some embodiments, the composition is a pharmaceutical composition.

In some embodiments, the composition may be in a form suitable foradministration by a route selected from the group consisting ofparenteral, transdermal, oral and topical. In some embodiments, thecomposition may be in a form suitable for administration by injection.In some embodiments, the composition is a parenteral formulation foradministration by a route selected from the group consisting ofsubcutaneous, intravenous, intramuscular, intradermal, intraperitoneal,intraarterial, intracerebral, intracerebroventricular, intraosseus andintrathecal.

In some embodiments, the subject to be treated with the compositionsdisclosed herein may be selected from the group of humans and non-humanmammals.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thefigures and by study of the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures. Dimensionsof components and features shown in the figures are generally chosen forconvenience and clarity of presentation and are not necessarily shown toscale. It is intended that the embodiments and figures disclosed hereinare to be considered illustrative rather than restrictive. The figuresare listed below.

FIG. 1 displays bar graphs showing the forelimb placement delta score inGroups A-F (Respective Entries nr. 4 (group-A); 27 (group B); 31 (GroupC); 2 (Group E); 26 (Group F) of Table 1A) of the forelimb-placing testin a tMCAO stroke model in rats. Group D is placebo control.

FIG. 2 displays bar graphs showing neurological scoring in Groups A-F(Respective Entries nr. 4 (group-A); 27 (group B); 31 (Group C); 2(Group E); 26 (Group F) of Table 1A) of the neurological score test in atMCAO stroke model in rats. Group D is placebo control.

FIGS. 3A and 3B display graphs showing the effect of “Combination A”(Table 1A, Entry nr. 25) on cytolysis induced by glutamate treatment,normalized on the 72 hours data point. FIG. 3A-shows cytolysis kineticcurves normalized on the 72 h data point (just before glutamatetreatment. FIG. 3B shows areas under curves (AUC) of the cytolysiskinetics calculated from the 72 h time point to the end of the kinetics.*: p<0.05, **: p<0.01 compared to the vehicle-treated group, One-WayANOVA followed by Dunnett's post hoc test.

FIGS. 4A and 4B display graphs showing the effect of “Combination B”(Table 1A, Entry nr. 31) on cytolysis induced by glutamate treatment,normalization on the 72 hours data point. FIG. 4A—shows cytolysiskinetic curves normalized on the 72 h data point (just before glutamatetreatment. FIG. 4B shows areas under curves (AUC) of the cytolysiskinetics calculated from the 72 h time point to the end of the kinetics.*: p<0.05, **: p<0.01 compared to the vehicle-treated group, One-WayANOVA followed by Dunnett's post hoc test.

FIG. 5A shows line graph displaying the effect of “Combination A” (Table1A, Entry nr. 25) on Okadaic acid (OKA) induced Tau hyperphosphorylationafter a 24 hours pre-treatment. *: p<0.05 compared to the Non Treatedcondition (NT). One Way ANOVA followed by Dunnett's multiple comparisontest.

FIG. 5B shows line graph displaying the effect of “Combination B” (Table1A, Entry nr. 31) on Okadaic acid (OKA) induced Tau hyperphosphorylationafter a 24 hours pre-treatment or as a co-treatment. *: p<0.05 comparedto the Non Treated condition (NT). One Way ANOVA followed by Dunnett'smultiple comparison test.

FIG. 6 displays the effect of “Combination A” (Table 1A, Entry nr. 25)and “Combination B” (Table 1A, Entry nr. 31) on choice reaction time.Reaction times to each two-lever presentation, expressed as the meanvalue per animal per session. Reaction times to each two-leverpresentation, expressed as the mean value per animal per session. (*):compared with Aged controls (vehicle s.c.).

FIG. 7 displays the effect of “Combination A” (Table 1A, Entry nr. 25)and “Combination B” (Table 1A, Entry nr. 31) on the duration of socialinvestigation at the second contact compared with the duration at thefirst contact for each group. The effect of combination A and B on theduration of social investigation at the second contact compared with theduration at the first contact for each group. (a): compared with firstcontact. (b): compared with Adult controls (vehicle s.c.). (c): comparedwith Aged controls (vehicle s.c.).

FIG. 8 displays the effect of “Combination A” (Table 1A, Entry nr. 25)and “Combination B” (Table 1A, Entry nr. 31) on the recognition index(=C2/C1). The effect of combination A and B on the recognition index(=C2/C1). (b): compared with Adult controls (vehicle s.c.). (c):compared with Aged controls (vehicle s.c.).

FIGS. 9A and 9B display effect of “Combination A” (Table 1A, Entry nr.25) treatment on 6-OH DA induced toxicity on mesencephalic primarycultures and TH positive neurons.

FIGS. 10A and 10B display the effect of “Combination B” (Table 1A, Entrynr. 31) treatment on 6-OH DA induced toxicity on mesencephalic primarycultures and TH positive neurons.

FIGS. 11A and 11B displays the effect of “Combination C” (Table 1A,Entry nr. 34) treatment on 6-OH DA induced toxicity on mesencephalicprimary cultures and TH positive neurons.

FIGS. 12A and 12B display the mean of “Combination A” (Table 1A, Entrynr. 25) and “Combination C” (Table 1A, Entry nr. 34) on Paw Placementdifferences (change from day 7).

FIGS. 13A and 13B display respectively the mean TH-positive area in thestriatum for “Combinations A, B and C” (Respective Entries 25, 31 and 34in Table 1A); and the normalized mean TH-positive area in the striatumfor “Combinations A, B and C” (Respective Entries 25, 31 and 34 in Table1A).

DETAILED DESCRIPTION OF THE INVENTION

As disclosed herein, it has been surprisingly found that combinations oftriterpenoic acids and neutral triterpenoid compounds show high activityin treating a condition selected from Alzheimer's disease (AD),Parkinson's Diseases (PD) and vascular dementia (VD).

It has further been surprisingly found that combinations of triterpenoicacids and neutral triterpenoid compounds show high activity in treatinga condition selected from Multi System Atrophy (MSA), ProgressiveSupranuclear Palsy (PSP) and tauopathic conditions and diseases.

Specific combinations of triterpenoic acids and neutral terpenoidcompounds surprisingly show enhanced therapeutic efficacy.

Definitions

As used herein the term “plurality” refers to more than one, preferablymore than two. As used herein the term “synergistic” means more thanadditive.

As used herein, the term “acid-base extraction” refers to a procedure inwhich an organic solvent solution containing organic acidic (typically,organic carboxylic acids) and organic non-acidic components istreated/extracted with one or more basic aqueous solution(s). As aresult, the organic acidic components are deprotonated and thusconverted into their corresponding deprotonated ionic salt forms(typically, organic anionic carboxylates) and as a result will dissolvein the said basic aqueous solution. The non-acidic organic componentswill not deprotonate, thus will stay behind in the original organicsolution phase. The deprotonated acids may also form an intermediateoily and/or emulsion layer, especially when multigram amounts are beingextracted. The basic aqueous solution containing the deprotonated saltforms of the acidic components, together with the oily and/or emulsionlayer (if present), is acidified, resulting in the reformation of theprotonated acid forms of the organic acidic components. These protonatedacid forms (acidic fraction) can be removed from the acidified aqueoussolution in several ways depending on the properties of the acidiccompounds. One option for removing the acidic fraction from theacidified solution is by reextraction into a suitable organic solvent.Examples 1A and 1B hereinbelow describe a non-limiting example of anacid-base extraction as described above. Depending on the solubility andphysical form of the acidic compounds (e.g. if the acidic fractiontypically comprises a separated/precipitated solid) in the acidifiedaqueous solution, the acidic fraction may be isolated via filtration ofthe acidified aqueous solution.

As stated above, the original organic solution phase remaining afterextraction with basic aqueous solution(s) contains the non-acidicorganic components. In the case of mastic gum these non-acidiccomponents consist of neutral triterpenoids and the mixture is referredto as a neutral fraction. Examples 1A and 1B below describe a particular(but non-limiting) method for the isolation of a certain acidic and acertain neutral fraction from mastic gum.

From the isolated acidic fraction and neutral fraction, the individualtriterpenoic acids and neutral triterpenoids can be isolated usingmethods known in the art such as column chromatography and HPLC. Severalreferences presented in the introduction of the current applicationcontain examples of separation methods for triterpenoic acids andneutral triterpenoids from mastic gum.

Instead of using a basic aqueous solution for the acid-base extraction,basic forms of ion-exchange resins can be used as well. In these cases,upon contact with the ion-exchange resin the acidic organic components(acidic fraction-typically, organic carboxylic acids) are captured intheir deprotonated anionic form (typically, organic anioniccarboxylates) by the resin. The resin is subsequently removed from theinitial solution, leaving non-acidic components behind. The acidiccomponents (acidic fraction) are subsequently released from the resin bytreatment of the resin with a suitable acidic solution. The use ofion-exchange resins for acid-base extractions is especially suitable forprocess scale up and can be used for the development of (semi)continuousextraction processes.

Examples of the above acid-base extractions and other variations can befound in many textbooks and other publications, and are consideredcommon knowledge to those skilled in the art. An example of a usefultextbook is “Vogel's Textbook of Practical Organic Chemistry”, 5^(th)Edition, 1989, (p. 162-163).

As used herein, the term “degree of purity” refers to the content of aspecified chemical compound in a preparation, expressed as a percentageon a weight per weight basis of the specified chemical compound relativeto other chemical compounds in the preparation.

As used herein, “terpene compounds” refers to isoprene-containinghydrocarbons, having isoprene units (CH₂C(CH₃)CHCH₂) in a head-to-tailorientation. Terpene hydrocarbons in general, have the molecular formula(C₅Hs)_(n), and include hemiterpenes, (C5), monoterpenes (C10),sesquiterpenes (C15), diterpenes (C20), triterpenes (C30), andtetraterpenes (C40) which respectively have 1, 2, 3, 4, 6 and 8 isopreneunits. Terpenes may be further classified as acyclic or cyclic.

As used herein, “terpenoids” and “terpenoid compounds” interchangeablyrefer to terpene-related compounds, which contain at least one oxygenatom in addition to isoprene units, and thus include alcohols,aldehydes, ketones, ethers, such as but not limited to, carboxylic acidsderivatives thereof, such as esters. Terpenoids are subdivided accordingto the number of carbon atoms in a manner similar to terpene and thusinclude hemiterpenoids, (C5), monoterpenoids (C10), sesquiterpenoids(C15), diterpenoids (C20), triterpenoids (C30), and tetraterpenoids(C40) which respectively have 1, 2, 3, 4, 6 and 8 isoprene units. Theskeleton of terpenoids may differ from strict additivity of isopreneunits by the loss or shift of a fragment, commonly a methyl group.Examples of monoterpenoids include camphor, eugenol, menthol andborneol. Examples of diterpenoids include phytol, retinol and taxol.Examples of triterpenoids include betulinic acid and lanosterol.Terpenoids may be acyclic or may contain one or more ring-structures.Triterpenoids may be acyclic or may contain one or more ring-structures.The rings may contain only carbon atoms, or alternatively may containone or more oxygen atoms besides carbon atoms. Common ring-sizes rangefrom three-membered rings to ten-membered rings. Larger ring sizes of upto at least twenty-membered rings are possible. More than one ring andmore than one ring-size maybe present in a single triterpenoid. In casea triterpenoid contains more than one ring, the rings may be present andseparated by one or more acyclic bonds; alternatively, the rings may bedirectly connected via connections of the annealed type, the bridgedtype, the spiro-type or combinations of any of these types. Multiplyannealed, fused, bridged, or spiro-type ringsystems are possible.Combinations of singly and multiply annealed, bridged, fused, spiro-typerings are possible. Combinations of isolated rings and connected ringsin the same triterpenoid are possible.

As used herein, “terpenoic acids” refer to terpenoid compoundscontaining at least one carboxylic acid functional group (COOH). Theterpenoic acids may additionally contain one or more otheroxygen-containing functional groups, for example, but not limited tohydroxyl, keto, aldehyde, ether (cyclic and non-cyclic), ester (cyclicand non-cyclic). They also may contain one or more C═C double bond, eachdouble bond may be of the cis, trans, E-type, Z-type, as well asmono-substituted, di-substituted, tri-substituted or tetrasubstituted(meaning no vinylic H-substituent), independently from other C═C bonds.The carboxylic acid group may be present in the protonated form (COOH)or in deprotonated anionic form (COO⁻).

As used herein, “triterpenoic acids” refer to triterpenoid compoundscontaining at least one carboxylic acid group. The triterpenoic acidsmay additionally contain one or more other oxygen-containing functionalgroups for example, but not limited to, hydroxyl, keto, aldehyde, ether(cyclic and non-cyclic) and ester (cyclic and non-cyclic). They also maycontain one or more C═C double bond, each double bond may be of the cis,trans, E- or Z-type, as well as monosubstituted, disubstituted,trisubstituted or tetrasubstituted (meaning no vinylic H-substituent),independently from other C═C bonds. The carboxylic acid group may bepresent in the protonated form (COOH) or in deprotonated anionic form(COO).

As used herein, “neutral terpenoids” refer to terpenoid compoundslacking a carboxylic acid group. The neutral triterpenoids may containone or more other oxygen-containing functional groups for example, butnot limited to, hydroxyl, keto, aldehyde, ether (cyclic and non-cyclic)and ester (cyclic and non-cyclic). They also may contain one or more C═Cdouble bond, each double bond may be of the cis, trans, E- or Z-type, aswell as monosubstituted, disubstituted, trisubstituted ortetrasubstituted (meaning no vinylic H-substituent), independently fromother C═C bonds.

As used herein, “neutral triterpenoids” refer to triterpenoid compoundslacking a carboxylic acid group. The neutral triterpenoids may containone or more other oxygen-containing functional groups for example, butnot limited to, hydroxyl, keto, aldehyde, ether (cyclic and non-cyclic)and ester (cyclic and non-cyclic). They also may contain one or more C═Cdouble bond, each double bond may be of the cis, trans, E- or Z-type, aswell as monosubstituted, disubstituted, trisubstituted ortetrasubstituted (meaning no vinylic H-substituent), independently fromother C═C bonds.

As used herein, “an oligomeric form of a terpenoic acid” refers to anoligomeric terpenoid acid in which the monomeric units are either of thesame terpenoic acid or of different terpenoic acids, and are joined inany possible arrangements, and are connected one to another through anypossible bond or functional group, such as a C—C bond, but not limitedto, an ester group or an ether group.

As used herein, “an oligomeric form of a triterpenoic acid” refers to anoligomeric triterpenoid acid in which the monomeric units are either ofthe same triterpenoic acid or of different triterpenoic acids, and arejoined in any possible arrangements, and are connected one to anotherthrough any possible bond or functional group, such as, but not limitedto, a C—C bond, an ester group or an ether group.

As used herein, the terms “mastic”, “mastic resin”, “gum mastic” and“mastic gum”, are used interchangeably to refer to a tree resin (alsoknown as an oleoresin) obtained as an exudate from any tree classifiedin the family Anacardiaceae. Trees in the genus Pistacia, most notablyPistacia lentiscus L., and in particular the cultivar P. lentiscus L.cv. Chia (cultivated on the Greek island of Chios), are known for theirhigh yield of gum mastic. Other varieties include P. lentiscus L. var.emarginata Engl., and P. lentiscus L. var. latifolia Coss. Additionalspecies of Pistacia include for example, P. atlantica, P. palestina, P.saportae, P. terebinthus, P. vera and P. integerrima.

As used herein, the terms “masticadienoic acid”, “masticadienonic acid”,“masticadienoic” and “masticadienonic” may interchangeably be used.

In order to provide clarity with respect to the molecular structure ofcompounds frequently mentioned and referred to in this application, alist of structures with names and acronyms used in this application ispresented below.

Masticadienonic acid refers to 24-Z-masticadienonic acid, the acronymMDA used in the current application refers to this compound. Thechemical structure of 24-Z-masticadienonic acid is as follows:

As used herein, the terms “isomasticadienoic acid”, “isomasticadienonicacid”, “isomasticadienoic” and “isomasticadienonic” may interchangeablybe used.

Isomasticadienonic acid refers 24-Z-isomasticadienonic acid, the acronymIMDA used in the current application refers to this compound. Thechemical structure of 24-Z-isomasticadienonic acid is as follows:

Oleanonic acid (OLN) has the following molecular structure:

Moronic acid (MO) has the following molecular structure:

24-Z-masticadienolic acid (MLA) has the following structure, the3-hydroxyl group has the beta-configuration:

24-Z-epimasticadienolic acid (epi-MLA) has the following structure, the3-hydroxyl group has the alpha-configuration:

24-Z-isomasticadienolic acid (IMLA) has the following structure, the3-hydroxyl group has the beta-configuration:

24-Z-epi-isomasticadienolic acid (epi-IMLA) has the following structure,the 3-hydroxyl group has the beta-configuration:

24-Z-3-O-acetyl-masticadienolic acid (3-OAc-MLA) has the followingmolecular structure:

24-Z-3-O-acetyl-epimasticadienolic acid (3-OAc-epi-MLA) has thefollowing molecular structure:

24-Z-3-O-acetyl-isomasticadienolic acid (3-OAc-IMLA) has the followingmolecular structure:

24-Z-3-O-acetyl-epiisomasticadienolic acid (3-OAc-epi-IMLA) has thefollowing molecular structure:

It is to be understood that in the context of this disclosure, in casethe “24-Z” is omitted from any the abovementioned compound names, it isthis particular 24-Z-isomer that is referred to.

The term “NF-1” is directed to the neutral triterpenoid compound(8R)-3-beta, 8-dihydroxypolypoda-13E,17E,21-triene (also referred to asMyrrhanol C), having the structure as set forth in scheme I:

The term “NF-2” is directed to the neutral triterpenoid compound((8R)-3-Oxo-8-hydroxypolypoda-13E,17E,21-triene, having the structure asset forth in scheme II:

The term “NF-3” is directed to the neutral triterpenoid compoundOleanonic aldehyde, having the structure as set forth in scheme III:

-   -   The term “NF-4” is directed to the neutral triterpenoid compound        Tirucallol (C-20 Epimer of Euphol), having the structure as set        forth in scheme IV:

-   -   The term “NF-A” is directed to the neutral triterpenoid compound        28-hydroxylup-20(29)-en-3-one (also referred to as Betulon),        having the structure as set forth in scheme V:

The term “NF-B” is directed to the neutral triterpenoid compound28-hydroxy-beta-amyrone (also referred to as Oleanonic alcohol), havingthe structure as set forth in scheme VI:

The term “NF-P” is directed to the neutral triterpenoid compound20-hydroxydammar-24-en-3-one (also referred to as Dipterocarpol), havingthe structure as set forth in scheme VII.

Additional neutral triterpenoids isolated from mastic gum neutralfraction are the following:

As used herein, the term “essential oil” refers to a volatile oilderived from the leaves, stem, flower or twigs of plants orsynthetically-made compounds that have the same chemical attributes. Theessential oil usually carries the odor or flavor of the plant. Eachplant essential oil or derivative thereof may be extracted from naturalsources or synthetically made. Chemically essential oils generallycontain mixtures of mono- and sesquiterpenes or corresponding mixturesof such terpenoids as major constituents, which have lower molecularweights in comparison with triterpenes and titerpenoids. Particularly,this group comprise saturated and unsaturated acyclic monoterpenes orsesquiterpenes including alcohol or aldehyde moieties, benzenoidaromatic compounds containing at least one oxygenated substituent orside chain, or a monocarbocyclic terpene generally having a six-memberedring bearing one or more oxygenated substituents. The mastic resincontains about 2-4% of such compounds. As used herein, “essential oil”further includes derivatives thereof, including racemic mixtures,enantiomers, diastereomers, hydrates, salts, solvates, metabolites,analogs, and homologs.

As used herein, “substantially devoid” means that a preparation orpharmaceutical composition according to the invention that generallycontains less than about 5% of the stated substance. For example, lessthan about 3%, less than 1%, less than 0.5%, less than 0.1%.

As used herein, the term “consisting essentially of” means that the onlyactive pharmaceutical ingredient in the formulation or method thattreats a specified condition is the specifically recited therapeuticingredient in the particular embodiment or claim. The presence of otheringredients, e.g., excipients and/or lubricants, etc., is not precluded.The presence of additional other pharmaceutically active agents is alsonot precluded, as long as the latter do not have actual effect on saidcondition.

As used herein, “therapeutically effective amount” refers to that amountof a pharmaceutical ingredient which substantially induces, promotes orresults in a desired therapeutic effect.

As used herein, “pharmaceutically acceptable carrier” refers to adiluent or vehicle, which is used to enhance the delivery and/orpharmacokinetic properties of a pharmaceutical ingredient with which itis formulated, but has no therapeutic effect of its own, nor does itinduce or cause any undesirable or untoward effect or adverse reactionin the subject.

As used herein, “pharmaceutically acceptable hydrophobic carrier” refersto a hydrophobic non-polar diluent or vehicle in which a composition isdissolved or suspended.

As used herein, “tauopathies” are a group of neurodegenerative diseasesassociated with pathological aggregation of so-called tau-protein ingliofibrillary or neurofibrillary tangles. Tau protein in its normalstate is highly soluble and belongs to the class ofmicrotubule-associated-proteins (MAPs) These tangles (also known as“paired helical filaments” are formed through hyperphosphorylation oftau-protein which results in aggregation into an insoluble form.Tauopathies can be divided into two groups, primary and secondarytauopathies. Non-limiting examples of primary tauopathies are Primaryage-related tauopathy (PART, also known as neurofibrillarytangle-predominant senile dementia; Dementia puglilistica (chronictraumatic encephalopathy); Progressive Supranuclear Palsy (PSP);Corticobasal degeneration; Frontotemporal dementia and Parkinsonismlinked to chromosome 17; Pick's disease; Ganglioma; Gangliocytoma;Meningoangiomatosis; Postencephalitic parkinsonism; Subacute SclerosingPanencephalitis.

As used herein, alpha-synucleinopathies (synucleinopathies) areneurodegenerative diseases characterized by the abnormal accumulation ofaggregates of the alpha-synuclein protein in neurons, nerve fibers orglial cells.

Parkinson's disease (PD) is a debilitating neurodegenerative disordercharacterized by the progressive loss of dopaminergic (DA) neurons inthe substantia nigra pars compacta (SNc), leading to a marked dopamine(DA) depletion in striatum, the primary projection region, as well asextrastriatal nuclei of the basal ganglia. PD has a characteristicclinical syndrome of bradykinesia, tremor, rigidity, and posturalinstability. There are a large number of different disorders that canhave some or all of these clinical features, and the clinical syndromeis referred to as “parkinsonism”. Disorders in which parkinsonism is aprominent part are referred to as “parkinsonian disorders.” PD is one ofa host of parkinsonian disorders. Degenerative parkinsonian disorderscan be inherited or sporadic, but are all characterized by neuronal lossin selective populations of vulnerable neurons. The common denominatorof all degenerative parkinsonian disorders is loss of dopaminergicneurons of the substantia nigra that project to the putamen (i.e.,dopaminergic nigrostriatal pathway). Parkinson-plus syndromes includesuch conditions as, multiple system atrophy (MSA), Progressivesupranuclear palsy (PSP), Parkinsonism-dementia-amyotrophic lateralsclerosis complex, Corticobasal ganglionic degeneration (CBD) andDementia with Lewy bodies (DLB).

As used herein the term “about” in reference to a numerical value statedherein is to be understood as the stated value+/−10%.

Compositions Comprising Triterpenoic Acids and Neutral Triterpenoids

In some embodiments, the present invention provides compositionscomprising or consisting of specific triterpenoic acids and neutraltriterpenoids, these compositions are shown to have an unexpectedsynergetic therapeutic effect in the treatment of Alzheimer's disease(AD), Parkinson's Diseases (PD) and/or vascular dementia (VD).

In some embodiments, the present invention provides compositionscomprising or consisting of specific triterpenoic acids and neutraltriterpenoids, these compositions are shown to have an unexpectedsynergetic therapeutic effect in the treatment of related conditions,such as, Multiple System Atrophy (MSA) and Progressive SupranuclearPalsy (PSP).

In some embodiments, the present invention provides compositionscomprising or consisting of specific triterpenoic acids and neutraltriterpenoids, these compositions are shown to have an unexpectedsynergetic therapeutic effect in the treatment of tauopathic diseasesand conditions.

The triterpenoic acids and neutral triterpenoid compounds may beobtained from a plant source, such as for example mastic gum, or may bethe products of chemical synthesis reactions. In some embodiments, anyone of the triterpenoic acids and neutral triterpenoids may be theproduct of a biochemical reaction or a product produced by a microbialorganism. In some embodiments, any one of the triterpenoic acids andneutral triterpenoids may be the product of a fermentation process. Insome embodiments, any one of the triterpenoic acids and neutraltriterpenoids may produced by a combination of a chemical synthesis anda biochemical reaction. In some embodiments, any one of the triterpenoicacids and neutral triterpenoids may be produced by a combination of achemical synthesis and a fermentation process. In some embodiments, anyone of the triterpenoic acids and neutral triterpenoids may be producedby a combination of any of the aboveindicated options. In case of abiochemical reaction or microbial process, the biochemical agent and themicrobial agent may be a naturally occurring agent or may be a modifiedagent not naturally occurring. Modification of these agents may beachieved using modern biochemical methods such as for example geneticengineering. Said biochemical agents and microbial agents not occurringnaturally may also be created using synthetic biology methods.

The current invention relates to the unexpected biological andpharmaceutical properties of the disclosed pharmaceutical compositionscomprising triterpenoic acid(s) and neutral triterpenoid(s). Thecombination of triterpenoic acid(s) and neutral triterpenoid(s) resultsin an overall pharmaceutical activity which cannot be obtained by usingonly the triterpenoic acids or only the neutral triterpenoids.

In some embodiments, the compositions may correspond to combinations ofcompounds in which some are chemically synthesized and some are derivedfrom plant sources.

In some embodiments, the compositions may correspond to combinations ofcompounds in which each compound may independently have been derivedfrom a plant source, or may be the product of a chemical synthesis, abiochemical reaction, or a microbial process (e.g. fermentation) asindicated above.

In some embodiments, the present invention provides compositionscomprising combinations comprising or consisting of at least onetriterpenoic acid and at least one neutral triterpenoid havingtherapeutic activity, as detailed herein. In some embodiments, thepresent invention provides compositions comprising combinationscomprising at least one triterpenoic acid and at least one neutraltriterpenoid having therapeutic activity, and a pharmaceuticallyacceptable carrier. In some embodiments, there is provided a compositioncomprising at least one triterpenoic acid, at least one neutraltriterpenoid and a pharmaceutically acceptable carrier.

In some embodiments, the triterpenoic acid may be selected from at leastone of masticadienonic acid (MDA), isomasticadienonic acid (IMDA),masticadienolic acid (MLA), isomasticadienolic acid (IMLA), 3-O-acetylmasticadienolic acid, 3-O-acetyl epimasticadienolic acid, 3-O-acetylisomasticadienolic acid, 3-O-acetyl epi-isomasticadienolic acid,oleanonic acid (OA) and moronic acid (MA), or any combination thereof.Each possibility is a separate embodiment.

In some embodiments, when MDA is one of the triterpenoic acids, MDA maycomprise about 2-80% of the total weight of the triterpenoic acids. Insome embodiments, MDA may comprise about 10-70% of the total weight ofthe triterpenoic acids. In some embodiments, MDA may comprise about15-60% of the total weight of the triterpenoic acids. In someembodiments, MDA may comprise about 20-50% of the total weight of thetriterpenoic acids. In some embodiments, MDA may comprise about 20-40%of the total weight of the triterpenoic acids. In some embodiments, MDAmay comprise about 40-50% of the total weight of the triterpenoic acids.In some embodiments, MDA may comprise about 50% of the total weight ofthe triterpenoic acids.

In some embodiments, when IMDA is one of the triterpenoic acids, IMDAmay comprise about 2-80% of the total weight of the triterpenoic acids.In some embodiments, IMDA may comprise about 10-70% of the total weightof the triterpenoic acids. In some embodiments, IMDA may comprise about15-60% of the total weight of the triterpenoic acids. In someembodiments, IMDA may comprise about 20-50% of the total weight of thetriterpenoic acids. In some embodiments, IMDA may comprise about 20-40%of the total weight of the triterpenoic acids. In some embodiments, IMDAmay comprise about 40-50% of the total weight of the triterpenoic acids.In some embodiments, IMDA may comprise about 50% of the total weight ofthe triterpenoic acids.

In some embodiments, when MLA is one of the triterpenoic acids, MLA maycomprise about 0-80% of the total weight of the triterpenoic acids. Insome embodiments, MLA may comprise about 0-70% of the total weight ofthe triterpenoic acids. In some embodiments, MLA may comprise about0-25% of the total weight of the triterpenoic acids. In someembodiments, MLA may comprise about 0-15% of the total weight of thetriterpenoic acids. In some embodiments, MLA may comprise about 8% ofthe total weight of the triterpenoic acids.

In some embodiments, when IMLA is one of the triterpenoic acids, IMLAmay comprise about 0-80% of the total weight of the triterpenoic acids.In some embodiments, IMLA may comprise about 0-70% of the total weightof the triterpenoic acids. In some embodiments, IMLA may comprise about0-25% of the total weight of the triterpenoic acids. In someembodiments, IMLA may comprise about 0-15% of the total weight of thetriterpenoic acids. In some embodiments, IMLA may comprise about 8% ofthe total weight of the triterpenoic acids.

In some embodiments, when MA is one of the triterpenoic acids, MA maycomprise about 0-80% of the total weight of the triterpenoic acids. Insome embodiments, MA may comprise about 0-70% of the total weight of thetriterpenoic acids. In some embodiments, MA may comprise about 0-40% ofthe total weight of the triterpenoic acids. In some embodiments, MA maycomprise about 0-30% of the total weight of the triterpenoic acids. Insome embodiments, MA may comprise about 5-20% of the total weight of thetriterpenoic acids. In some embodiments, MA may comprise about 12-15% ofthe total weight of the triterpenoic acids.

In some embodiments, when OA is one of the triterpenoic acids, OA maycomprise about 0-80% of the total weight of the triterpenoic acids. Insome embodiments, OA may comprise about 0-70% of the total weight of thetriterpenoic acids. In some embodiments, OA may comprise about 0-50% ofthe total weight of the triterpenoic acids. In some embodiments, OA maycomprise about 5-35% of the total weight of the triterpenoic acids. Insome embodiments, OA may comprise about 10-25% of the total weight ofthe triterpenoic acids. In some embodiments, MA may comprise about18-20% of the total weight of the triterpenoic acids.

In some embodiments, when 3-O-acetyl masticadienolic acid is one of thetriterpenoic acids, 3-O-acetyl masticadienolic acid may comprise about0-80% of the total weight of the triterpenoic acids. In someembodiments, 3-O-acetyl masticadienolic acid may comprise about 0-70% ofthe total weight of the triterpenoic acids. In some embodiments,3-O-acetyl masticadienolic acid may comprise about 0-25% of the totalweight of the triterpenoic acids. In some embodiments, 3-O-acetylmasticadienolic acid may comprise about 0-15% of the total weight of thetriterpenoic acids. In some embodiments, 3-O-acetyl masticadienolic acidmay comprise about 4-7% of the total weight of the triterpenoic acids.

In some embodiments, when 3-O-acetyl isomasticadienolic acid is one ofthe triterpenoic acids, 3-O-acetyl isomasticadienolic acid may compriseabout 0-80% of the total weight of the triterpenoic acids. In someembodiments, 3-O-acetyl isomasticadienolic acid may comprise about 0-70%of the total weight of the triterpenoic acids. In some embodiments,3-O-acetyl isomasticadienolic acid may comprise about 0-25% of the totalweight of the triterpenoic acids. In some embodiments, 3-O-acetylisomasticadienolic acid may comprise about 0-15% of the total weight ofthe triterpenoic acids. In some embodiments, 3-O-acetylisomasticadienolic acid may comprise about 4-7% of the total weight ofthe triterpenoic acids.

In some embodiments, when 3-O-acetyl-epimasticadienolic acid is one ofthe triterpenoic acids, 3-O-acetyl masticadienolic acid may compriseabout 0-80% of the total weight of the triterpenoic acids. In someembodiments, 3-O-acetyl epimasticadienolic acid may comprise about 0-70%of the total weight of the triterpenoic acids. In some embodiments,3-O-acetyl masticadienolic acid may comprise about 0-25% of the totalweight of the triterpenoic acids. In some embodiments, 3-O-acetylepimasticadienolic acid may comprise about 0-15% of the total weight ofthe triterpenoic acids. In some embodiments, 3-O-acetylepimasticadienolic acid may comprise about 4-7% of the total weight ofthe triterpenoic acids.

In some embodiments, when 3-O-acetyl epiisomasticadienolic acid is oneof the triterpenoic acids, 3-O-acetyl epiisomasticadienolic acid maycomprise about 0-80% of the total weight of the triterpenoic acids. Insome embodiments, 3-O-acetyl epiisomasticadienolic acid may compriseabout 0-70% of the total weight of the triterpenoic acids. In someembodiments, 3-O-acetyl epiisomasticadienolic acid may comprise about0-25% of the total weight of the triterpenoic acids. In someembodiments, 3-O-Acetyl epiisomasticadienolic acid may comprise about0-15% of the total weight of the triterpenoic acids. In someembodiments, 3-O-Acetyl epimasticadienolic acid may comprise about 4-7%of the total weight of the triterpenoic acids.

In some embodiments, the neutral triterpenoid may be selected from atleast one of (8R)-3-beta, 8-dihydroxypolypoda-13E,17E,21-triene(8-dihydroxypolypoda-13E,17E,21-triene; NF-1),(8R)-3-Oxo-8-hydroxypolypoda-13E,17E,21-triene (NF-2), Oleanonicaldehyde (NF-3), Tirucallol (NF-4), 28-hydroxylup-20(29)-en-3-one(NF-A), 28-hydroxy-beta-amyrone (NF-B), 20-hydroxydammar-24-en-3-one(NF-P), 3-beta-hydroxy-13-alpha-malabarica-14(26),17E,21-triene,20-hydroxy-lupan-3-one, 28-Nor-17-hydroxylupen-3-one,28-oxo-lupen-3-one, 28-nor-beta-amyrone, Isomasticadienonic aldehyde,Isomasticadienediol, Oleanolic aldehyde (28-oxo-beta-amyrin),3-beta-20-dihydroxylupane, Masticadienonic aldehyde,3-oxo-malabarica-14(26),17E,21-triene, Beta-amyrone, Beta-amyrin,Germanicol, or any combination thereof. Each possibility is a separateembodiment.

In some embodiments, the neutral triterpenoid may be selected from atleast one of (8R)-3-beta, 8-dihydroxypolypoda-13E,17E,21-triene(8-dihydroxypolypoda-13E,17E,21-triene; NF-1),(8R)-3-Oxo-8-hydroxypolypoda-13E,17E,21-triene (NF-2), Oleanonicaldehyde (NF-3), Tirucallol (NF-4), 28-hydroxylup-20(29)-en-3-one(NF-A), 28-hydroxy-beta-amyrone (NF-B), 20-hydroxydammar-24-en-3-one(NF-P), 3-beta-hydroxy-13-alpha-malabarica-14(26),17E,21-triene,20-hydroxy-lupan-3-one, 28-Nor-17-hydroxylupen-3-one,28-oxo-lupen-3-one, 28-nor-beta-amyrone, Isomasticadienonic aldehyde,Isomasticadienediol, Masticadienediol, Oleanolic aldehyde(28-oxo-beta-amyrin), 3-beta-20-dihydroxylupane, Masticadienonicaldehyde, 3-oxo-malabarica-14(26),17E,21-triene, Beta-amyrone,Beta-amyrin, Germanicol, or any combination thereof. Each possibility isa separate embodiment.

In some embodiments, the neutral triterpenoid may be selected from atleast one of (8R)-3-beta, 8-dihydroxypolypoda-13E,17E,21-triene(8-dihydroxypolypoda-13E,17E,21-triene; NF-1),(8R)-3-Oxo-8-hydroxypolypoda-13E,17E,21-triene (NF-2), Oleanonicaldehyde (NF-3), Tirucallol (NF-4), 28-hydroxylup-20(29)-en-3-one(NF-A), 28-hydroxy-beta-amyrone (NF-B),3-beta-hydroxy-13-alpha-malabarica-14(26),17E,21-triene,20-hydroxy-lupan-3-one, 28-Nor-17-hydroxylupen-3-one,28-oxo-lupen-3-one, 28-nor-beta-amyrone, Isomasticadienonic aldehyde,Isomasticadienediol, Oleanolic aldehyde (28-oxo-beta-amyrin),3-beta-20-dihydroxylupane, Masticadienonic aldehyde,3-oxo-malabarica-14(26),17E,21-triene, Beta-amyrone, Beta-amyrin,Germanicol, or any combination thereof. Each possibility is a separateembodiment.

In some embodiments, the neutral triterpenoid may be selected from atleast one of (8R)-3-beta, 8-dihydroxypolypoda-13E,17E,21-triene(8-dihydroxypolypoda-13E,17E,21-triene; NF-1),(8R)-3-Oxo-8-hydroxypolypoda-13E,17E,21-triene (NF-2), Oleanonicaldehyde (NF-3), Tirucallol (NF-4), 28-hydroxylup-20(29)-en-3-one(NF-A), 28-hydroxy-beta-amyrone (NF-B),3-beta-hydroxy-13-alpha-malabarica-14(26),17E,21-triene,20-hydroxy-lupan-3-one, 28-Nor-17-hydroxylupen-3-one,28-oxo-lupen-3-one, 28-nor-beta-amyrone, Isomasticadienonic aldehyde,Isomasticadienediol, Masticadienediol, Oleanolic aldehyde(28-oxo-beta-amyrin), 3-beta-20-dihydroxylupane, Masticadienonicaldehyde, 3-oxo-malabarica-14(26),17E,21-triene, Beta-amyrone,Beta-amyrin, Germanicol, or any combination thereof. Each possibility isa separate embodiment.

In some embodiments, when NF-1 is one of the neutral triterpenoids, theamount of NF-1 with respect to the total amount of neutral triterpenoidsmay be in the range of about 0% to about 80%. In some embodiments, theamount of NF-1 with respect to the total amount of neutral triterpenoidsmay be in the range of about 0% to about 50%. In some embodiments, theamount of NF-1 with respect to the total amount of neutral triterpenoidsmay be in the range of about 5% to about 25%. In some embodiments, theamount of NF-1 with respect to the total amount of neutral triterpenoidsmay be in the range of about 9% to about 13%.

In some embodiments, when NF-2 is one of the neutral triterpenoids, theamount of NF-2 with respect to the total amount of neutral triterpenoidsmay be in the range of about 0% to about 80%. In some embodiments, theamount of NF-2 with respect to the total amount of neutral triterpenoidsmay be in the range of about 0% to about 50%. In some embodiments, theamount of NF-2 with respect to the total amount of neutral triterpenoidsmay be in the range of about 5% to about 25%. In some embodiments, theamount of NF-2 with respect to the total amount of neutral triterpenoidsmay be in the range of about 9% to about 13%.

In some embodiments, when NF-3 is one of the neutral triterpenoids, theamount of NF-3 with respect to the total amount of neutral triterpenoidsmay be in the range of about 0% to about 80%. In some embodiments, theamount of NF-3 with respect to the total amount of neutral triterpenoidsmay be in the range of about 0% to about 50%. In some embodiments, theamount of NF-3 with respect to the total amount of neutral triterpenoidsmay be in the range of about 5% to about 25%. In some embodiments, theamount of NF-3 with respect to the total amount of neutral triterpenoidsmay be in the range of about 9% to about 13%.

In some embodiments, when NF-4 is one of the neutral triterpenoids, theamount of NF-4 with respect to the total amount of neutral triterpenoidsmay be in the range of about 0% to about 80%. In some embodiments, theamount of NF-4 with respect to the total amount of neutral triterpenoidsmay be in the range of about 0% to about 50%. In some embodiments, theamount of NF-4 with respect to the total amount of neutral triterpenoidsmay be in the range of about 5% to about 25%. In some embodiments, theamount of NF-4 with respect to the total amount of neutral triterpenoidsmay be in the range of about 9% to about 13%.

In some embodiments, when NF-P is one of the neutral triterpenoids, theamount of NF-P with respect to the total amount of neutral triterpenoidsmay be in the range of about 0% to about 50%. In some embodiments, theamount of NF-P with respect to the total amount of neutral triterpenoidsmay be in the range of about 0% to about 25%. In some embodiments, theamount of NF-P with respect to the total amount of neutral triterpenoidsmay be in the range of about 0% to about 7%. In some embodiments, theamount of NF-P with respect to the total amount of neutral triterpenoidsmay be in the range of about 6% to about 7%.

In some embodiments, when NF-A is one of the neutral triterpenoids, theamount of NF-A with respect to the total amount of neutral triterpenoidsmay be in the range of about 0% to about 25%. In some embodiments, theamount of NF-A with respect to the total amount of neutral triterpenoidsmay be in the range of about 0% to about 15%. In some embodiments, theamount of NF-A with respect to the total amount of neutral triterpenoidsmay be in the range of about 0% to about 6%. In some embodiments, theamount of NF-A with respect to the total amount of neutral triterpenoidsmay be in the range of about 4% to about 6%.

In some embodiments, when NF-B is one of the neutral triterpenoids, theamount of NF-B with respect to the total amount of neutral triterpenoidsmay be in the range of about 0% to about 25%. In some embodiments, theamount of NF-B with respect to the total amount of neutral triterpenoidsmay be in the range of about 0% to about 15%. In some embodiments, theamount of NF-B with respect to the total amount of neutral triterpenoidsmay be in the range of about 0% to about 6%. In some embodiments, theamount of NF-B with respect to the total amount of neutral triterpenoidsmay be in the range of about 4% to about 6%.

In some embodiments, the triterpenoic acids may comprise from about 1%to about 80% of the total active ingredients of the composition. In someembodiments, the triterpenoic acids may comprise from about 10% to about80% of the total active ingredients of the composition. In someembodiments, the triterpenoic acids may comprise from about 20% to about80% of the total active ingredients of the composition. In someembodiments, the triterpenoic acids may comprise from about 30% to about70% of the total active ingredients of the composition. In someembodiments, the triterpenoic acids may comprise from about 35% to about65% of the total active ingredients of the composition. In someembodiments, the triterpenoic acids may comprise from about 40% to about60% of the total active ingredients of the composition.

In some embodiments, the triterpenoic acids may comprise from about0.01% to about 80% of the total composition. In some embodiments, thetriterpenoic acids may comprise from about 0.01% to about 50% of thetotal composition. In some embodiments, the triterpenoic acids maycomprise from about 0.01% to about 10% of the total composition. In someembodiments, the triterpenoic acids may comprise from about 0.1% toabout 10% of the total composition. In some embodiments, thetriterpenoic acids may comprise from about 0.5% to about 4% of the totalcomposition. In some embodiments, the triterpenoic acids may comprisefrom about 1% to about 3.5% of the total composition. In someembodiments, the triterpenoic acids may comprise from about 1.5% toabout 3% of the total composition. In some embodiments, the triterpenoicacids may comprise from about 1.75% to about 2.75% of the totalcomposition. In some embodiments, the triterpenoic acids may comprisefrom about 2% to about 2.5% of the total composition.

In some embodiments, the neutral triterpenoids may comprise from about1% to about 80% of the total active ingredients of the composition. Insome embodiments, the neutral triterpenoids may comprise from about 10%to about 80% of the total active ingredients of the composition. In someembodiments, the neutral triterpenoids may comprise from about 20% toabout 80% of the total active ingredients of the composition. In someembodiments, the neutral triterpenoids may comprise from about 30% toabout 70% of the total active ingredients of the composition. In someembodiments, the neutral triterpenoids may comprise from about 35% toabout 65% of the total active ingredients of the composition. In someembodiments, the neutral triterpenoids may comprise from about 40% toabout 60% of the total active ingredients of the composition.

In some embodiments, the neutral triterpenoids may comprise from about0.01% to about 80% of the total composition. In some embodiments, theneutral triterpenoids may comprise from about 0.01% to about 50% of thetotal composition. In some embodiments, the neutral triterpenoids maycomprise from about 0.01% to about 10% of the total composition. In someembodiments, the neutral triterpenoids may comprise from about 0.1% toabout 10% of the total composition. In some embodiments, the neutraltriterpenoids may comprise from about 0.5% to about 4% of the totalcomposition. In some embodiments, the neutral triterpenoids may comprisefrom about 1% to about 3.5% of the total composition. In someembodiments, the neutral triterpenoids may comprise from about 1.5% toabout 3% of the total composition. In some embodiments, the neutraltriterpenoids may comprise from about 1.75% to about 2.75% of the totalcomposition. In some embodiments, the neutral triterpenoids may comprisefrom about 2% to about 2.5% of the total composition.

In some embodiments, the combinations comprise at least one of MDA andIMDA as the triterpenoic acids and at least one of NF-1 and NF-2 as theneutral triterpenoids.

In some embodiments, the combinations may include at least one of MDAand IMDA as the triterpenoic acids and at least one of NF-1 and NF-2 asthe neutral triterpenoids.

In some embodiments, the combinations may include at least MDA as thetriterpenoic acid and at least NF-1 as the neutral triterpenoid.

In some embodiments, the combinations may include at least MDA as thetriterpenoic acid and at least NF-2 as the neutral triterpenoid.

In some embodiments, the combinations may include at least IMDA as thetriterpenoic acid and at least NF-1 as the neutral triterpenoid.

In some embodiments, the combinations may include at least IMDA as thetriterpenoic acid and at least NF-2 as the neutral triterpenoid.

In some embodiments, the combinations may include at least MDA and IMDAas the triterpenoic acids and at least NF-1 and NF-2 as the neutraltriterpenoids.

In some embodiments, the combinations may include at least MDA and IMDAas the triterpenoic acids and at least NF-1, NF-2, NF-3 and NF-4 as theneutral triterpenoids. In some embodiments, the combinations may includeat least MDA and IMDA as the triterpenoic acids and at least NF-1, NF-2,NF-3, NF-4, NF-A, NF—B, NF-P as the neutral triterpenoids. In someembodiments, the combinations may include at least MDA and IMDA as thetriterpenoic acids and at least NF-1, NF-2, NF-3, NF-4, NF-A, and NF-Bas the neutral triterpenoids. In some embodiments, the combinations mayinclude at least MDA, MLA, IMDA and IMLA as the triterpenoic acids andat least NF-1, NF-2, NF-3 and NF-4 as the neutral triterpenoids. In someembodiments, the combinations may include at least MDA, MLA, IMDA andIMLA as the triterpenoic acids and at least NF-1, NF-2, NF-3, NF-4,NF-A, NF—B and NF-P as the neutral triterpenoids. In some embodiments,the combinations may include at least MDA, MLA, IMDA and IMLA as thetriterpenoic acids and at least NF-1, NF-2, NF-3, NF-4, NF-A and NF-B asthe neutral triterpenoids. Such compositions unexpectedly exhibit asynergistic effect, whereby the combination of compounds exhibit amarkedly improved therapeutic effect in the treatment of conditions,such as Alzheimer's disease (AD), Parkinson's Diseases (PD) and/orvascular dementia (VD).

In some embodiments, the neutral triterpenoid comprises at least NF-1and at least one additional neutral triterpenoid. In some embodiments,the neutral triterpenoid comprises at least NF-2 and at least oneadditional neutral triterpenoid. In some embodiments, the neutraltriterpenoid comprises at least NF-1, NF-2 and at least one additionalneutral triterpenoid. In some embodiments, the additional neutraltriterpenoid is selected from the group consisting of NF-1, NF-2, NF-3,NF-4, NF-A, NF—B and NF-P. In some embodiments, the additional neutraltriterpenoid is selected from the group consisting of NF-1, NF-2, NF-3,NF-4, NF-A and NF-B. Each possibility is a separate embodiment. In someembodiments, the additional neutral triterpenoid is selected from thegroup consisting of NF-2, NF-3, NF-4, NF-A, NF-B, and NF-P. In someembodiments, the additional neutral triterpenoid is selected from thegroup consisting of NF-2, NF-3, NF-4, NF-A and NF-B. In someembodiments, the additional neutral triterpenoid is selected from thegroup consisting of NF-1, NF-3, NF-4, NF-A, NF—B and NF-P. In someembodiments, the additional neutral triterpenoid is selected from thegroup consisting of NF-1, NF-3, NF-4, NF-A and NF-B. In someembodiments, the additional neutral triterpenoid is selected from thegroup consisting of NF-3, NF-4, NF-A, NF—B and NF-P. In someembodiments, the additional neutral triterpenoid is selected from thegroup consisting of NF-3, NF-4, NF-A and NF-B. In some embodiments, theadditional neutral triterpenoid is selected from NF-3 and TirucallolNF-4. Various combinations of some of these compounds exhibit anunexpected synergistic effect in the treatment of Alzheimer's disease(AD), Parkinson's Diseases (PD) and/or vascular dementia (VD). Variouscombinations of some of these compounds exhibit an unexpectedsynergistic effect in the treatment of conditions, such as, MultipleSystem Atrophy (MSA) and Progressive Supranuclear Palsy (PSP). Variouscombinations of some of these compounds exhibit an unexpectedsynergistic effect in the treatment of various tauopathic diseases andconditions.

In some embodiments, there is provided a composition comprising at leastone of MDA and IMDA as the triterpenoic acid and at least one of NF-1and NF-2 as the neutral triterpenoid.

In some embodiments, there is provided a composition comprising at leastone of MDA, IMDA, MLA, IMLA, 3-O-acetyl masticadienolic acid, 3-O-acetylepimasticadienolic acid, 3-O-acetyl isomasticadienolic acid, 3-O-acetylepi-isomasticadienolic acid, OA and MA, in addition to at least one ofNF-1, NF-2, NF-3, NF-4, NF-A, NF—B and NF-P. Each possibility is aseparate embodiment of the invention.

In some embodiments, there is provided a composition comprising at leastone of MDA, IMDA, MLA, IMLA, 3-O-acetyl masticadienolic acid, 3-O-acetylepimasticadienolic acid, 3-O-acetyl isomasticadienolic acid, 3-O-acetylepi-isomasticadienolic acid, OA and MA, in addition to at least one ofNF-1, NF-2, NF-3, NF-4, NF-A and NF-B. Each possibility is a separateembodiment of the invention.

In some embodiments, the composition comprises at least one ofmasticadienonic acid MDA, IMDA, MLA, IMLA, 3-O-acetyl masticadienolicacid, 3-O-acetyl isomasticadienolic acid, OA and MA; in addition to atleast one of NF-1, NF-2, NF-3 and NF-4. In some embodiments, thecomposition comprises at least one of MDA and IMDA; in addition to atleast one of NF-1, NF-2, NF-3 and NF-4. In some embodiments, thecomposition comprises at least one of MDA and IMDA; in addition to atleast one of NF-1, NF-2, NF-3, NF-4, NF-A, NF—B and NF-P. In someembodiments, the composition comprises at least one of MDA and IMDA; inaddition to at least one of NF-1, NF-2, NF-3, NF-4, NF-A and NF-B. Insome embodiments the composition may further include a pharmaceuticallyacceptable carrier. Each possibility is a separate embodiment of theinvention.

In some embodiments, there is provided a composition comprising at leastone of NF-1 and NF-2, in addition to at least one of MDA, IMDA, MLA,IMLA, 3-O-acetyl masticadienolic acid, 3-O-acetyl epimasticadienolicacid, 3-O-acetyl isomasticadienolic acid, 3-O-acetylepi-isomasticadienolic acid, OA and MA; and at least one of NF-3, NF-4,NF-A, NF—B and NF-P.

In some embodiments, there is provided a composition comprising at leastone of NF-1 and NF-2, in addition to at least one of MDA, IMDA, MLA,IMLA, 3-O-acetyl masticadienolic acid, 3-O-acetyl epimasticadienolicacid, 3-O-acetyl isomasticadienolic acid, 3-O-acetylepi-isomasticadienolic acid, OA and MA; and at least one of NF-3, NF-4,NF-A and NF-B.

In some embodiments, there is provided a composition comprising NF-1,NF-2, at least one of MDA, IMDA, MLA, IMLA, 3-O-acetyl masticadienolicacid, 3-O-acetyl epimasticadienolic acid, 3-O-acetyl isomasticadienolicacid, 3-O-acetyl epi-isomasticadienolic acid; OA, MA, and at least oneof NF-3, NF-4, NF-A, NF—B and NF-P.

In some embodiments, there is provided a composition comprising NF-1,NF-2, at least one of MDA, IMDA, MLA, IMLA, 3-O-acetyl masticadienolicacid, 3-O-acetyl epimasticadienolic acid, 3-O-acetyl isomasticadienolicacid, 3-O-acetyl epi-isomasticadienolic acid; OA, MA, and at least oneof NF-3, NF-4, NF-A and NF-B.

In some embodiments, there is provided a composition comprising at leastone of NF-1 and NF-2, in addition to at least one of MDA, IMDA, MLA,IMLA, 3-O-acetyl masticadienolic acid, 3-O-acetyl isomasticadienolicacid, OA and MA, and at least one of NF-3 and NF-4.

In some embodiments, there is provided a composition comprising NF-1 andNF-2, in addition to at least one of masticadienonic acid MDA, IMDA,MLA, IMLA, 3-O-acetyl masticadienolic acid, 3-O-acetylisomasticadienolic acid, OA and MA, and at least one of NF-3 and NF-4.

In some embodiments, there is provided a composition comprising at leastone of NF-1 and NF-2 in addition to at least one of MDA, IMDA, MLA andIMLA, and at least one of NF-3, NF-4, NF-A, NF—B and NF-P.

In some embodiments, there is provided a composition comprising at leastone of NF-1 and NF-2 in addition to at least one of MDA, IMDA, MLA andIMLA, and at least one of NF-3, NF-4, NF-A and NF-B.

In some embodiments, there is provided a composition comprising NF-1 andNF-2, in addition to at least one of MDA, IMDA, MLA and IMLA, and atleast one of NF-3, NF-4, NF-A, NF—B and NF-P.

In some embodiments, there is provided a composition comprising NF-1 andNF-2, in addition to at least one of MDA, IMDA, MLA and IMLA, and atleast one of NF-3, NF-4, NF-A and NF-B.

In some embodiments, there is provided a composition comprising at leastone of NF-1 and NF-2, in addition to at least one of masticadienonicacid MDA and IMDA; and at least one of NF-3 and NF-4.

In some embodiments, there is provided a composition comprising NF-1 andNF-2, in addition to at least one of MDA and IMDA; and at least one ofNF-3 and NF-4.

In some embodiments, there is provided a composition comprising at leastone of NF-1 and NF-2, in addition to at least one of MDA, IMDA, MLA,IMLA, 3-O-acetyl masticadienolic acid, 3-O-acetyl isomasticadienolicacid, OA and MA, and at least one of NF-3, NF-4, NF-A, NF—B and NF-P.

In some embodiments, there is provided a composition comprising at leastone of NF-1 and NF-2, in addition to at least one of MDA, IMDA, MLA,IMLA, 3-O-acetyl masticadienolic acid, 3-O-acetyl isomasticadienolicacid, OA and MA, and at least one of NF-3, NF-4, NF-A and NF-B.

In some embodiments, there is provided a composition comprising NF-1 andNF-2, in addition to at least one of MDA, IMDA, MLA, IMLA, 3-O-acetylmasticadienolic acid, 3-O-acetyl isomasticadienolic acid, OA and MA, andat least one of NF-3, NF-4, NF-A, NF—B and NF-P.

In some embodiments, there is provided a composition comprising NF-1 andNF-2, in addition to at least one of MDA, IMDA, MLA, IMLA, 3-O-acetylmasticadienolic acid, 3-O-acetyl isomasticadienolic acid, OA and MA, andat least one of NF-3, NF-4, NF-A and NF-B.

In some embodiments, there is provided a composition comprising at leastone of NF-1 and NF-2, in addition to at least one of masticadienonicacid MDA and IMDA; and at least one of NF-3, NF-4, NF-A, NF—B and NF-P.

In some embodiments, there is provided a composition comprising at leastone of NF-1 and NF-2, in addition to at least one of masticadienonicacid MDA and IMDA; and at least one of NF-3, NF-4, NF-A and NF-B.

In some embodiments, there is provided a composition comprising NF-1 andNF-2, in addition to at least one of MDA and IMDA and at least one ofNF-3, NF-4, NF-A, NF—B and NF-P.

In some embodiments, there is provided a composition comprising NF-1 andNF-2, in addition to at least one of MDA and IMDA and at least one ofNF-3, NF-4, NF-A and NF-B.

In some embodiments, there is provided a composition comprising NF-1 andNF-2, in addition to at least one of MDA and IMDA.

In some embodiments, the composition may include not more than 15triterpenoids. In some embodiments, the composition may include not morethan 14 triterpenoids. In some embodiments, the composition may includenot more than 13 triterpenoids. In some embodiments, the composition mayinclude not more than 12 triterpenoids. In some embodiments, thecomposition may include not more than 11 triterpenoids. In someembodiments, the composition may include not more than 10 triterpenoids.In some embodiments, the composition may include not more than 9triterpenoids. In some embodiments, the composition may include not morethan 8 triterpenoids. In some embodiments, the composition may includenot more than 7 triterpenoids. In some embodiments, the composition mayinclude not more than 6 triterpenoids.

In some embodiments, there is provided a combination comprising at leastone of MDA, IMDA, MLA, IMLA, 3-O-acetyl masticadienolic acid, 3-O-acetylepimasticadienolic acid, 3-O-acetyl isomasticadienolic acid, 3-O-acetylepi-isomasticadienolic acid; OA and MA; in addition to at least one ofNF-1, NF-2, NF-3, NF-4, NF-A, NF—B and NF-P. Each possibility is aseparate embodiment of the invention. In some embodiments, there isprovided a combination comprising at least one of MDA, IMDA, MLA, IMLA,3-O-acetyl masticadienolic acid, 3-O-acetyl epimasticadienolic acid,3-O-acetyl isomasticadienolic acid, 3-O-acetyl epi-isomasticadienolicacid; OA and MA; in addition to at least one of NF-1, NF-2, NF-3, NF-4,NF-A and NF-B. Each possibility is a separate embodiment of theinvention.

In some embodiments, the combination comprises at least one of MDA,IMDA, MLA, IMLA, 3-O-acetyl masticadienolic acid, 3-O-acetylisomasticadienolic acid, OA and MA; in addition to at least one of NF-1,NF-2, NF-3 and NF-4. In some embodiments, the combination comprises atleast one of MDA and IMDA; in addition to at least one of (NF-1, NF-2,NF-3 and NF-4. In some embodiments, the combination comprises at leastone of MDA and IMDA; in addition to at least one of NF-1, NF-2, NF-3,NF-4, NF-A, NF—B and NF-P. In some embodiments, the combinationcomprises at least one of MDA and IMDA; in addition to at least one ofNF-1, NF-2, NF-3, NF-4, NF-A and NF-B. In some embodiments, thecombination may further include a pharmaceutically acceptable carrier.

In some embodiments, the composition further comprises at least oneneutral triterpenoid selected from the group consisting of:3-beta-hydroxy-13-alpha-malabarica-14(26),17E,21-triene,20-hydroxy-lupan-3-one, 28-Nor-17-hydroxylupen-3-one,28-oxo-lupen-3-one, 28-nor-beta-amyrone, Isomasticadienonic aldehyde,Isomasticadienediol, Oleanolic aldehyde (28-oxo-beta-amyrin),3-beta-20-dihydroxylupane, Masticadienonic aldehyde,3-oxo-malabarica-14(26),17E,21-triene. Each possibility is a separateembodiment of the invention.

In some embodiments, the composition further comprises at least oneneutral triterpenoid selected from the group consisting of:3-beta-hydroxy-13-alpha-malabarica-14(26),17E,21-triene,20-hydroxy-lupan-3-one, 28-Nor-17-hydroxylupen-3-one,28-oxo-lupen-3-one, 28-nor-beta-amyrone, Isomasticadienonic aldehyde,Isomasticadienediol, Masticadienediol, Oleanolic aldehyde(28-oxo-beta-amyrin), 3-beta-20-dihydroxylupane, Masticadienonicaldehyde, 3-oxo-malabarica-14(26),17E,21-triene. Each possibility is aseparate embodiment of the invention.

In some embodiments, in a composition comprising more than onetriterpenoic acid, and if present in such composition, IMDA and MDA arepresent at a ratio of about 1:1 w/w.

In some embodiments, in a composition comprising more than onetriterpenoic acid, and if present in such composition, MDA, IMDA, MLAIMLA are present at a ratio of about 1:1:0.2:0.2 (5:5:1:1) w/wrespectively.

In some embodiments, if present in such composition IMDA, MDA, NF-1,NF-2, NF-3 and NF-4 are present at a ratio of about 1:1:0.5:0.5:0.5:0.33(6:6:3:3:3:2) w/w respectively.

In some embodiments, if present in such composition IMDA, MDA, NF-1,NF-2, NF-3, NF-4, NF—P, NF-A, and NF—B are present at a ratio of about1:1:0.5:0.5:0.5:0.33:0.33:0.25:0.25 (12:12:6:6:6:4:4:3:3) w/wrespectively.

In some embodiments, if present in such composition IMDA, MDA, NF-1,NF-2, NF-3, NF-4, NF-A, and NF—B are present at a ratio of about1:1:0.5:0.5:0.5:0.33:0.25:0.25 (12:12:6:6:6:4:4:3:3) w/w respectively.

In some embodiments, if present in such composition, IMDA, MDA, NF-1 andNF-2 are present at a ratio of about 1:1:0.5:0.5 (2:2:1:1) w/wrespectively.

In some embodiments, if present in such composition, IMDA, MDA, OA,3-O-acetyl-masticadienolic acid, 3-O-acetyl-isomasticadienolic acid,NF-1 and NF-2 are present at a ratio of about2:2:0.75:0.75:1.5:1:1:1:0.67 w/w respectively.

In some embodiments, if present in such composition, NF-1, NF-2, NF-3and NF-4 present at a ratio of about 1:1:1:0.67 (3:3:3:2) w/wrespectively.

In some embodiments, if present in such composition, NF-1, NF-2, NF-3,NF-4, NF—P, NF-A, and NF—B are present at a ratio of about1:1:1:0.67:0.67:0.5:0.5 (6:6:6:4:4:3:3) respectively.

In some embodiments, if present in such composition, NF-1, NF-2, NF-3,NF-4, NF-A, and NF—B are present at a ratio of about 1:1:1:0.67:0.5:0.5(6:6:6:4:4:3:3) respectively.

In some embodiments, the composition further comprises at least oneneutral triterpenoid selected from the group consisting of:Beta-amyrone, Beta-amyrin and Germanicol. Each possibility is a separateembodiment of the invention.

In some embodiments, the combination may further comprise at least onetriterpenoic acid selected from the group consisting of: oleanolic acid,ursonic acid and ursolic acid. Each possibility is a separate embodimentof the invention.

In some embodiments, the triterpenoic acid(s) may be obtained from aplant source. In some embodiments, any one of the triterpenoic acids maybe obtained from a plant source. In some embodiments, at least onetriterpenoic acid may be obtained from a plant source. In someembodiments, the neutral triterpenoid(s) may be obtained from a plantsource. In some embodiments, any one of the neutral triterpenoids may beobtained from a plant source. In some embodiments, at least one neutraltriterpenoid may be obtained from a plant source. In some embodiments,the plant source may include mastic gum.

In some embodiments, any one of the triterpenoic acids and/or theneutral triterpenoid may be isolated from a natural source or may be theproduct of a chemical synthesis. In some embodiments, the triterpenoicacids and/or the neutral triterpenoids may be isolated from a naturalsource or may be the product of a chemical synthesis.

In some embodiments, any one of the triterpenoic acids and neutraltriterpenoids may be the product of a biochemical reaction or a productproduced by a microbial organism. In some embodiments, any one of thetriterpenoic acids and neutral triterpenoids may be the product of afermentation process. In some embodiments, any one of the triterpenoicacids and neutral triterpenoids may produced by a combination of achemical synthesis and a biochemical reaction. In some embodiments, anyone of the triterpenoic acids and neutral triterpenoids may be producedby a combination of a chemical synthesis and a fermentation process. Insome embodiments, the any one of triterpenoic acids and neutraltriterpenoids may be produced by a combination of any of theaboveindicated options. In case of a biochemical reaction or microbialprocess, the biochemical agent and the microbial agent my be a naturallyoccurring agent or may be a modified agent not naturally occurring.Modification of these agents may have been achieved using modernbiochemical methods such as for example genetic engineering. Saidbiochemical agents and microbial agents not occurring naturally may alsohave been created using synthetic biology methods.

In some embodiments, obtaining from a natural source may includeisolating from a natural source. In some embodiments, the isolation fromthe natural source may include isolation as individual compound(s) or asa group(s) of compounds. In some embodiments, the natural source mayinclude a plant material selected from the group consisting of a resin,a gum, leaves, twigs, roots, flowers, seeds, buds, bark, nuts and roots.Each possibility is a separate embodiment. In some embodiments, thenatural source may include a resin extracted from at least one plant. Insome embodiments, the natural source may include mastic gum.

In some embodiments, the natural source may include at least one plant.In some embodiments, the plant may be classified in the familyAnacardiaceae. In some embodiments, the plant may comprise at least oneplant classified in the genus/genera Pistacia and/or Schinus. In someembodiments, Pistacia may include species selected from the groupconsisting of P. lentiscus, P. lentiscus Latifolia Coss, P. lentiscusvar. Chia, P. atlantica, P. palestina, P. saportae, P. terebinthus, P.vera P. integerrima, P and. lentiscus L. Each possibility is a separateembodiment. In some embodiments, Pistacia may include the speciesPistacia lentiscus L. In some embodiments, Schinus may include thespecies S. molle. In some embodiments, the Pistacia may include thespecies Pistacia Lentiscus var. Chia.

In some embodiments, the triterpenoids may be obtained by a processcomprising or consisting one or more of the steps of:

-   -   (a) treating mastic gum with a polar organic solvent;    -   (b) isolating a fraction soluble in said polar organic solvent;    -   (c) optionally removing said polar organic solvent;    -   (d) treating the soluble fraction obtained in step (b) or (c)        with a non-polar organic solvent;    -   (e) isolating a fraction soluble in said non-polar organic        solvent;    -   (f) optionally removing said non-polar organic solvent;    -   (g) dissolving the fraction obtained in step (f) in a first        organic solvent;    -   (h) treatment of the solution obtained in step (g) or (e) with a        basic aqueous solution so as to obtain a basic aqueous fraction        containing triterpenoic acids in a deprotonated salt form and an        intermediate oily or emulsion phase in addition to the first        organic solution containing neutral triterpenoids;    -   (i) separating said basic aqueous fraction and the intermediate        oily/emulsion phase from the first organic solution    -   (j) acidifying the basic aqueous fraction and emulsion obtained        in step (i) with an acid;    -   (k) extracting the acidified fraction obtained in step (j) with        a second organic solvent;    -   (l) optionally contacting the organic fraction obtained in        step (k) with a drying agent;    -   (m) removing the second organic solvent, the drying agent and/or        excess acid from the fraction obtained in any of steps (j), (k)        or (l) thus providing an isolated acidic fraction;    -   (n) taking the first organic solution from step (i), optionally        contacting it with a drying agent; and    -   (o) removing the first organic solvent and the drying agent thus        providing an isolated neutral fraction.

The individual triterpenoic acids can be obtained by chromatographicseparation from the isolated acidic fraction obtained in step (m). Theindividual neutral triterpenoids can be obtained by chromatographicseparation from the isolated neutral fraction obtained in step (o).

The individually obtained triterpenoic acids and neutral triterpenoidsmay then be mixed as required in order to obtain the desiredpharmaceutical compositions.

In some embodiments, there is provided a composition comprising acombination of at least one triterpenoic acid and at least one neutraltriterpenoid, and a pharmaceutically acceptable carrier, wherein thetriterpenoic acid is selected from MDA, IMDA or both, wherein theneutral triterpenoid is selected from NF-1, NF-2, or both.

In some embodiments, the composition comprises MDA. In some embodiments,the composition comprises IMDA. In some embodiments, the compositioncomprises MDA and IMDA.

In some embodiments the composition further comprises at least oneadditional triterpenoic acid. In some embodiments, the additionaltriterpenoic acid is selected from the group consisting of MLA, IMLA,3-O-acetyl masticadienolic acid, 3-O-acetyl epimasticadienolic acid,3-O-acetyl isomasticadienolic acid, 3-O-acetyl epi-isomasticadienolicacid, OA, MA and combinations thereof.

In some embodiments, the composition comprises at least two additionaltriterpenoic acids. In some embodiments, the composition comprises atleast three additional triterpenoic acids. In some embodiments, thecomposition comprises at least four additional triterpenoic acids.

In some embodiments, the composition further comprises at least oneadditional neutral triterpenoid. In some embodiments, the additionalneutral triterpenoid is selected from the group consisting of NF-3,NF-4, NF-A, NF—B, NF—P and combinations thereof.

In some embodiments, the composition comprises at least two additionalneutral triterpenoids. In some embodiments, the composition comprises atleast three additional neutral triterpenoids. In some embodiments, thecomposition comprises at least four additional neutral triterpenoids.

In some embodiments, the composition further comprises at least oneadditional neutral triterpenoid. In some embodiments, the additionalneutral triterpenoid is selected from the group consisting of NF-3,NF-4, NF-A, NF—B and combinations thereof.

In some embodiments, the composition further comprises NF-P. In someembodiments, at least one of the additional neutral triterpenoids isselected from NF-3, NF-4 or both.

In some embodiments, the current invention provides a pharmaceuticalcomposition consisting essentially of MDA, IMDA, NF-1, NF-2, NF-3 andNF-4 as the pharmaceutically active ingredients; and a pharmaceuticallyacceptable carrier. In some embodiments, the current invention providesa pharmaceutical composition consisting essentially of MDA, IMDA, MLA,IMLA, NF-1, NF-2, NF-3, NF-4, NF-A, NF—B and NF-P as thepharmaceutically active ingredients; and a pharmaceutically acceptablecarrier. In some embodiments, the current invention provides apharmaceutical composition consisting essentially of MDA, IMDA, MLA,IMLA, NF-1, NF-2, NF-3, NF-4, NF-A and NF-B as the pharmaceuticallyactive ingredients; and a pharmaceutically acceptable carrier. In someembodiments, the current invention provides a pharmaceutical compositionconsisting essentially of MDA, IMDA, NF-1, NF-2, NF-3, NF-4, NF-A, NF—Band NF-P as the pharmaceutically active ingredients; and apharmaceutically acceptable carrier. In some embodiments, the currentinvention provides a pharmaceutical composition consisting essentiallyof MDA, IMDA, NF-1, NF-2, NF-3, NF-4, NF-A and NF-B as thepharmaceutically active ingredients; and a pharmaceutically acceptablecarrier. In some embodiments, the current invention provides apharmaceutical composition consisting essentially of MA, OA, MDA, IMDA3-O-acetyl masticadienolic acid, 3-O-acetyl isomasticadienolic acid,MLA, IMLA, NF-1, NF-2, NF-3 and NF-4 as the pharmaceutically activeingredients; and a pharmaceutically acceptable carrier. In someembodiments, the current invention provides a pharmaceutical compositionconsisting essentially of MA, OA, MDA, IMDA 3-O-acetyl masticadienolicacid, 3-O-acetyl isomasticadienolic acid, MLA, IMLA, NF-1, NF-2, NF-3,NF-4, NF-A, NF—B and NF-P as the pharmaceutically active ingredients;and a pharmaceutically acceptable carrier. In some embodiments, thecurrent invention provides a pharmaceutical composition consistingessentially of MA, OA, MDA, IMDA 3-O-acetyl masticadienolic acid,3-O-acetyl isomasticadienolic acid, MLA, IMLA, NF-1, NF-2, NF-3, NF-4,NF-A and NF-B as the pharmaceutically active ingredients; and apharmaceutically acceptable carrier. In some embodiments, there isprovided a pharmaceutical composition comprising pharmaceutically activeingredients consisting essentially of MA, OA, MDA, IMDA, 3-O-acetylmasticadienolic acid, 3-O-acetyl isomasticadienolic acid, NF-1, NF-2,NF-3, NF-4, NF-A and NF-B; and a pharmaceutically acceptable carrier. Insome embodiments, there is provided a pharmaceutical compositioncomprising pharmaceutically active ingredients consisting essentially ofMA, OA, MDA, IMDA, 3-O-acetyl masticadienolic acid, 3-O-acetylisomasticadienolic acid, NF-1, NF-2, NF-3 and NF-4; and apharmaceutically acceptable carrier. In some embodiments, there isprovided a pharmaceutical composition comprising pharmaceutically activeingredients consisting essentially of OA, MDA, IMDA, 3-O-acetylmasticadienolic acid, 3-O-acetyl isomasticadienolic acid, NF-1, NF-2,NF-3, NF-4, NF-A and NF-B; and a pharmaceutically acceptable carrier. Insome embodiments, there is provided a pharmaceutical compositioncomprising pharmaceutically active ingredients consisting essentially ofOA, MDA, IMDA, 3-O-acetyl masticadienolic acid, 3-O-acetylisomasticadienolic acid, NF-1, NF-2, NF-3 and NF-4; and apharmaceutically acceptable carrier. In some embodiments, there isprovided a pharmaceutical composition comprising pharmaceutically activeingredients consisting essentially of MDA, IMDA, NF-1, NF-2; and apharmaceutically acceptable carrier. In some embodiments, there isprovided a pharmaceutical composition comprising pharmaceutically activeingredients consisting essentially of MDA, IMDA, NF-1, NF-2, NF-3, NF-4,NF-A and NF-B as the sole pharmaceutically active ingredients; and apharmaceutically acceptable carrier. In some embodiments, there isprovided a pharmaceutical composition comprising pharmaceutically activeingredients consisting essentially of MDA, IMDA, NF-1, NF-2, NF-3 andNF-4 as the sole pharmaceutically active ingredients; and apharmaceutically acceptable carrier. In some embodiments, there isprovided a pharmaceutical composition comprising pharmaceutically activeingredients consisting essentially of MDA, IMDA, NF-1 and NF-2 as thesole pharmaceutically active ingredients; and a pharmaceuticallyacceptable carrier.

Any one of the triterpenoic acids, neutral triterpenoids, additionaltriterpenoic acids and/or the additional neutral triterpenoids may beisolated from a natural source such as mastic gum, or may be the productof a chemical synthesis.

In some embodiments, any one of MDA, IMDA, MLA, IMLA, 3-O-acetylmasticadienolic acid, 3-O-acetyl epimasticadienolic acid, 3-O-acetylisomasticadienolic acid, 3-O-acetyl epi-isomasticadienolic acid, OA, MA,NF-1, NF-2, NF-3, NF-4, NF-A, NF-B), and NF—P may be a product of achemical synthesis. In some embodiments, any one of the at least onetriterpenoic acid and the at least one neutral triterpenoid may be aproduct of a chemical synthesis.

In some embodiments, any one of MDA, IMDA, MLA, IMLA, 3-O-acetylmasticadienolic acid, 3-O-acetyl epimasticadienolic acid, 3-O-acetylisomasticadienolic acid, 3-O-acetyl epi-isomasticadienolic acid, OA, MA,NF-1, NF-2, NF-3, NF-4, NF-A and NF—B may be a product of a chemicalsynthesis. In some embodiments, any one of the at least one triterpenoicacid and the at least one neutral triterpenoid may be a product of achemical synthesis.

Plant species useful for obtaining the compositions of the inventioninclude without limitation, those of the genus Pistacia. Useful speciesof Pistacia include without limitation, P. lentiscus, P. lentiscuslatifolius Coss., P. lentiscus var. Chia, P. atlantica, P. palestina, P.saportae, P. terebinthus, P. vera and P. integerrima.

Analytical methods for determining the precise chemical structure of thetriterpenoic acids and neutral triterpenoids include nuclear magneticresonance (for example ¹H-NMR and ¹³C-NMR), various mass spectrometrymethods (for example MALDI-TOF), HPLC, combination methods such asLiquid Chromatography-Mass spectrometry (LC-MS; LC-MS/MS, UV-VISspectrometry, IR and FT-IR spectrometry and other methods as are knownin the art.

In some embodiments, the composition includes at least one triterpenoicacid and at least one neutral triterpenoid. In some embodiments, thetriterpenoic acid may include masticadienonic acid (MDA),isomasticadienonic acid (IMDA), masticadienolic acid (MLA),isomasticadienolic acid (IMLA), 3-O-acetyl masticadienolic acid,3-O-acetyl epimasticadienolic acid, 3-O-acetyl isomasticadienolic acid,3-O-acetyl epi-isomasticadienolic acid, oleanonic acid (OA), moronicacid (MA), or any combination thereof. In some embodiments, the neutraltriterpenoid may include 8-dihydroxypolypoda-13E,17E,21-triene (NF-1),(8R)-3-Oxo-8-hydroxypolypoda-13E, 17E,21-triene (NF-2), Oleanonicaldehyde (NF-3), Tirucallol (NF-4), 28-hydroxylup-20(29)-en-3-one(NF-A), 28-hydroxy-beta-amyrone (NF-B), 20-hydroxydammar-24-en-3-one(NF-P) or any combination thereof.

In some embodiments, the composition includes at least one triterpenoicacid and at least one neutral triterpenoid. In some embodiments, the atleast one triterpenoic acid may include masticadienonic acid (MDA),isomasticadienonic acid (IMDA), masticadienolic acid (MLA),isomasticadienolic acid (IMLA), 3-O-acetyl masticadienolic acid,3-O-acetyl epimasticadienolic acid, 3-O-acetyl isomasticadienolic acid,3-O-acetyl epi-isomasticadienolic acid, oleanonic acid (OA), moronicacid (MA), or any combination thereof. In some embodiments, the neutraltriterpenoid may include 8-dihydroxypolypoda-13E, 17E,21-triene (NF-1),(8R)-3-Oxo-8-hydroxypolypoda-13E, 17E,21-triene (NF-2), Oleanonicaldehyde (NF-3), Tirucallol (NF-4), 28-hydroxylup-20(29)-en-3-one(NF-A), 28-hydroxy-beta-amyrone (NF-B) or any combination thereof.

In some embodiments, the at least one triterpenoic acid is or consistsof masticadienonic acid (MDA). In some embodiments, the at least onetriterpenoic acid is or consists of isomasticadienonic acid (IMDA). Insome embodiments, the at least one triterpenoic acid may consist ofmasticadienonic acid (MDA) and isomasticadienonic acid (IMDA). In someembodiments, the triterpenoic acid may include at least masticadienonicacid (MDA) and isomasticadienonic acid (IMDA). In some embodiments, thetriterpenoic acid may include at least masticadienonic acid (MDA),isomasticadienonic acid (IMDA), masticadienolic acid (MLA) andisomasticadienolic acid (IMLA).

In some embodiments, the at least one neutral triterpenoid is orconsists of NF-1. In some embodiments, the at least one neutraltriterpenoid is or consists of NF-2. In some embodiments, the at leastone neutral triterpenoid may consist of NF-1 and NF-2. In someembodiments, the at least one neutral triterpenoid may include at leastNF-1 and NF-2. In some embodiments, the at least one neutraltriterpenoid may include at least NF-1, NF-2, NF-3 and NF-4. In someembodiments, the at least one neutral triterpenoid may include at leastNF-1, NF-2, NF-3, NF-4, NF-A and NF-B. In some embodiments, the at leastone neutral triterpenoid may include at least NF-1, NF-2, NF-3, NF-4,NF-A, NF—B and NF-P.

In some embodiments, the triterpenoic acid may include at leastmasticadienonic acid (MDA) and isomasticadienonic acid (IMDA). In someembodiments, the triterpenoic acid may include at least masticadienonicacid (MDA), isomasticadienonic acid (IMDA), masticadienolic acid (MLA)and isomasticadienolic acid (IMLA). In some embodiments, thetriterpenoic acid may include at least masticadienonic acid (MDA),isomasticadienonic acid (IMDA), 3-O-acetyl masticadienolic acid(3-OAc-MLA) and 3-O-acetyl isomasticadienolic acid (3-OAc-IMLA). In someembodiments, the triterpenoic acid may include at least masticadienonicacid (MDA), isomasticadienonic acid (IMDA), 3-O-acetylepimasticadienolic acid (3-OAc-epi-MLA) and 3-O-acetylepiisomasticadienolic acid (3-OAc-epi-IMLA). In some embodiments, thetriterpenoic acid may include at least masticadienonic acid (MDA),isomasticadienonic acid (IMDA), 3-O-acetyl masticadienolic acid(3-OAc-MLA), 3-O-acetyl isomasticadienolic acid (3-OAc-IMLA), 3-O-acetylepimasticadienolic acid (3-OAc-epi-MLA) and 3-O-acetylepiisomasticadienolic acid (3-OAc-epi-IMLA). In some embodiments,neutral triterpenoid may include at least NF-1 and NF-2. In someembodiments, the neutral triterpenoid may include at least NF-1, NF-2,NF-3 and NF-4. In some embodiments, the neutral triterpenoid may includeat least NF-1, NF-2, NF-3, NF-4, NF-A and NF-B. In some embodiments, theneutral triterpenoid may include at least NF-1, NF-2 and NF-4. In someembodiments, the neutral triterpenoid may include at least NF-2, NF-3and NF-4. In some embodiments, the neutral triterpenoid may include atleast NF-1, NF-2 and NF-3. In some embodiments, the neutral triterpenoidmay include at least NF-1, NF-3 and NF-4. In some embodiments, theneutral triterpenoid may include at least NF-1 and NF-3. In someembodiments, the neutral triterpenoid may include at least NF-1 andNF-4. In some embodiments, the neutral triterpenoid may include at leastNF-2 and NF-3. In some embodiments, the neutral triterpenoid may includeat least NF-2 and NF-4. In some embodiments, the neutral triterpenoidmay include at least NF-1, NF-2, NF-A and NF-B. In some embodiments, theneutral triterpenoid may include at least NF-1, NF-A and NF-B. In someembodiments, the neutral triterpenoid may include at least NF-2, NF-Aand NF-B. In some embodiments, the neutral triterpenoid may include atleast NF-1 and NF-A. In some embodiments, the neutral triterpenoid mayinclude at least NF-2 and NF-A. In some embodiments, the neutraltriterpenoid may include at least NF-1 and NF-B. In some embodiments,the neutral triterpenoid may include at least NF-2 and NF-B. In someembodiments, the neutral triterpenoid may include at least NF-1, NF-2and NF-A. In some embodiments, the neutral triterpenoid may include atleast NF-1, NF-2 and NF-B.

In some embodiments, the neutral triterpenoids consist essentially ofnot more than seven neutral triterpenoids. In some embodiments, theneutral triterpenoids consist essentially of not more than six neutraltriterpenoids. In some embodiments, the neutral triterpenoids consistessentially of not more than five neutral triterpenoids. In someembodiments, the neutral triterpenoids consist essentially of not morethan four neutral triterpenoids.

In some embodiments, the additional neutral triterpenoids consistessentially of not more than six neutral triterpenoids. In someembodiments, the additional neutral triterpenoids consist essentially ofnot more than five neutral triterpenoids. In some embodiments, theadditional neutral triterpenoids consist essentially of not more thanfour neutral triterpenoids.

In some embodiments, the additional neutral triterpenoids consistessentially of not more than three neutral triterpenoids. In someembodiments, the additional neutral triterpenoids consist essentially ofnot more than two neutral triterpenoids.

In some embodiments, the triterpenoic acids consists essentially of notmore than eight triterpenoic acids. In some embodiments, thetriterpenoic acids consists essentially of not more than seventriterpenoic acids. In some embodiments, the triterpenoic acids consistsessentially of not more than six triterpenoic acids. In someembodiments, the triterpenoic acids consists essentially of not morethan five triterpenoic acids. In some embodiments, the triterpenoicacids consists essentially of not more than four triterpenoic acids. Insome embodiments, the triterpenoic acids consists essentially of notmore than three triterpenoic acids. In some embodiments, thetriterpenoic acids consists essentially of not more than twotriterpenoic acids.

In some embodiments, the composition consists essentially of not morethan 15 triterpenoids. In some embodiments, the composition consistsessentially of not more than 14 triterpenoids. In some embodiments, thecomposition consists essentially of not more than 13 triterpenoids. Insome embodiments, the composition consists essentially of not more than12 triterpenoids. In some embodiments, the composition consistsessentially of not more than 11 triterpenoids. In some embodiments, thecomposition consists essentially of not more than 10 triterpenoids. Insome embodiments, the composition consists essentially of not more than9 triterpenoids. In some embodiments, the composition consistsessentially of not more than 8 triterpenoids. In some embodiments, thecomposition consists essentially of not more than 7 triterpenoids. Insome embodiments, the composition consists essentially of not more than6 triterpenoids.

The compositions disclosed herein unexpectedly exhibit a synergisticeffect, whereby the combination of compounds exhibit a markedly improvedtherapeutic effect in the treatment of Alzheimer's disease (AD),Parkinson's Diseases (PD) and/or vascular dementia (VD). Suchcombinations of compounds further exhibit an unexpected synergisticeffect in the treatment of Multiple System Atrophy (MSA) and ProgressiveSupranuclear Palsy (PSP) and tauopathic diseases and conditions.

In some embodiments, the composition comprises at least two triterpenoicacids selected from MDA, IMDA, MLA, IMLA, OA, MA, 3-O-acetylmasticadienolic acid, 3-O-acetyl epimasticadienolic acid, 3-O-acetylisomasticadienolic acid and 3-O-acetyl epi-isomasticadienolic acid. Insome embodiments, the composition comprises at least two triterpenoicacids selected from MDA, IMDA, MLA, IMLA, OA, MA, 3-O-acetylmasticadienolic acid and 3-O-acetyl isomasticadienolic acid. In someembodiments, the composition comprises at least two triterpenoic acidsselected from MDA, IMDA, MLA and IMLA. In some embodiments, thecomposition comprises at least two triterpenoic acids selected from MDA,IMDA, and MLA. In some embodiments, the composition comprises at leasttwo triterpenoic acids selected from MDA, IMDA and IMLA. In someembodiments, the composition comprises at least MDA and IMDA.

In some embodiments, the composition comprises at least threetriterpenoic acids selected from MDA, IMDA, MLA, IMLA, OA, MA,3-O-acetyl masticadienolic acid, 3-O-acetyl epimasticadienolic acid,3-O-acetyl isomasticadienolic acid and 3-O-acetyl epi-isomasticadienolicacid. In some embodiments, the composition comprises at least threetriterpenoic acids selected from MDA, IMDA, MLA, IMLA, OA, MA,3-O-acetyl masticadienolic acid and 3-O-acetyl isomasticadienolic acid.In some embodiments, the composition comprises at least threetriterpenoic acids selected from MDA, IMDA, MLA and IMLA. In someembodiments, the composition comprises at least MDA, IMDA, and MLA. Insome embodiments, the composition comprises at least MDA, IMDA and IMLA.

In some embodiments, the composition comprises at least fourtriterpenoic acids selected from MDA, IMDA, MLA, IMLA, OA, MA,3-O-acetyl masticadienolic acid, 3-O-acetyl epimasticadienolic acid,3-O-acetyl isomasticadienolic acid and 3-O-acetyl epi-isomasticadienolicacid. In some embodiments, the composition comprises at least fourtriterpenoic acids selected from MDA, IMDA, MLA, IMLA, OA, MA,3-O-acetyl masticadienolic acid and 3-O-acetyl isomasticadienolic acid.In some embodiments, the composition comprises at least MDA, IMDA, MLAand IMLA.

In some embodiments, the composition comprises at least two neutraltriterpenoids selected from NF-1, NF-2, NF-3, NF-4, NF-A, NF—B, NF-P,3-beta-hydroxy-13-alpha-malabarica-14(26),17E,21-triene,20-hydroxy-lupan-3-one, 28-Nor-17-hydroxylupen-3-one,28-oxo-lupen-3-one, 28-nor-beta-amyrone, Isomasticadienonic aldehyde,Isomasticadienediol, Oleanolic aldehyde (28-oxo-beta-amyrin),3-beta-20-dihydroxylupane, Masticadienonic aldehyde,3-oxo-malabarica-14(26),17E,21-triene, Beta-amyrone, Beta-amyrin andGermanicol. In some embodiments, the composition comprises at least twoneutral triterpenoids selected from NF-1, NF-2, NF-3, NF-4, NF-A, NF-B,3-beta-hydroxy-13-alpha-malabarica-14(26), 17E,21-triene,20-hydroxy-lupan-3-one, 28-Nor-17-hydroxylupen-3-one,28-oxo-lupen-3-one, 28-nor-beta-amyrone, Isomasticadienonic aldehyde,Isomasticadienediol, Masticadienediol, Oleanolic aldehyde(28-oxo-beta-amyrin), 3-beta-20-dihydroxylupane, Masticadienonicaldehyde, 3-oxo-malabarica-14(26),17E,21-triene, Beta-amyrone,Beta-amyrin and Germanicol. Each possibility is a separate embodiment.In some embodiments, the composition comprises at least two neutraltriterpenoids selected from NF-1, NF-2, NF-3, NF-4, NF-A, NF—B and NF-P.In some embodiments, the composition comprises at least two neutraltriterpenoids selected from NF-1, NF-2, NF-3, NF-4, NF-A and NF-B. Insome embodiments, the composition comprises at least two neutraltriterpenoids selected from NF-1, NF-2, NF-3 and NF-4. In someembodiments, the composition comprises at least two neutraltriterpenoids selected from NF-1, NF-2 and NF-3. In some embodiments,the composition comprises at least two neutral triterpenoids selectedfrom NF-1, NF-2 and NF-4. In some embodiments, the composition comprisesat least NF-1 and NF-2.

In some embodiments, the composition comprises at least three neutraltriterpenoids selected from NF-1, NF-2, NF-3, NF-4, NF-A, NF—B, NF-P,3-beta-hydroxy-13-alpha-malabarica-14(26),17E,21-triene,20-hydroxy-lupan-3-one, 28-Nor-17-hydroxylupen-3-one,28-oxo-lupen-3-one, 28-nor-beta-amyrone, Isomasticadienonic aldehyde,Isomasticadienediol, Oleanolic aldehyde (28-oxo-beta-amyrin),3-beta-20-dihydroxylupane, Masticadienonic aldehyde,3-oxo-malabarica-14(26),17E,21-triene, Beta-amyrone, Beta-amyrin andGermanicol. In some embodiments, the composition comprises at leastthree neutral triterpenoids selected from NF-1, NF-2, NF-3, NF-4, NF-A,NF-B, 3-beta-hydroxy-13-alpha-malabarica-14(26), 17E,21-triene,20-hydroxy-lupan-3-one, 28-Nor-17-hydroxylupen-3-one,28-oxo-lupen-3-one, 28-nor-beta-amyrone, Isomasticadienonic aldehyde,Isomasticadienediol, Masticadienediol, Oleanolic aldehyde(28-oxo-beta-amyrin), 3-beta-20-dihydroxylupane, Masticadienonicaldehyde, 3-oxo-malabarica-14(26),17E,21-triene, Beta-amyrone,Beta-amyrin and Germanicol. Each possibility is a separate embodiment.In some embodiments, the composition comprises at least three neutraltriterpenoids selected from NF-1, NF-2, NF-3, NF-4, NF-A, NF—B and NF-P.In some embodiments, the composition comprises at least three neutraltriterpenoids selected from NF-1, NF-2, NF-3, NF-4, NF-A and NF-B. Insome embodiments, the composition comprises at least three neutraltriterpenoids selected from NF-1, NF-2, NF-3 and NF-4. In someembodiments, the composition comprises at least NF-1, NF-2 and NF-3. Insome embodiments, the composition comprises at least NF-1, NF-2 andNF-4. In some embodiments, the composition comprises at least NF-1, NF-3and NF-4. In some embodiments, the composition comprises at least NF-3,NF-3 and NF-4.

In some embodiments, the composition comprises at least four neutraltriterpenoids selected from NF-1, NF-2, NF-3, NF-4, NF-A, NF—B, NF-P,3-beta-hydroxy-13-alpha-malabarica-14(26),17E,21-triene,20-hydroxy-lupan-3-one, 28-Nor-17-hydroxylupen-3-one,28-oxo-lupen-3-one, 28-nor-beta-amyrone, Isomasticadienonic aldehyde,Isomasticadienediol, Masticadienediol, Oleanolic aldehyde(28-oxo-beta-amyrin), 3-beta-20-dihydroxylupane, Masticadienonicaldehyde, 3-oxo-malabarica-14(26),17E,21-triene, Beta-amyrone,Beta-amyrin and Germanicol. Each possibility is a separate embodiment.In some embodiments, the composition comprises at least four neutraltriterpenoids selected from NF-1, NF-2, NF-3, NF-4, NF-A, NF-B,3-beta-hydroxy-13-alpha-malabarica-14(26),17E,21-triene,20-hydroxy-lupan-3-one, 28-Nor-17-hydroxylupen-3-one,28-oxo-lupen-3-one, 28-nor-beta-amyrone, Isomasticadienonic aldehyde,Isomasticadienediol, Masticadienediol, Oleanolic aldehyde(28-oxo-beta-amyrin), 3-beta-20-dihydroxylupane, Masticadienonicaldehyde, 3-oxo-malabarica-14(26),17E,21-triene, Beta-amyrone,Beta-amyrin and Germanicol. Each possibility is a separate embodiment.In some embodiments, the composition comprises at least four neutraltriterpenoids selected from NF-1, NF-2, NF-3, NF-4, NF-A and NF-B. Insome embodiments, the composition comprises at least four neutraltriterpenoids selected from NF-1, NF-2, NF-3, NF-4, NF-A, NF—B and NF-P.In some embodiments, the composition comprises at least NF-1, NF-2, NF-3and NF-4.

In some embodiments, combinations of triterpenoic acids and neutraltriterpenoids may be substantially devoid of essential oils.

In some embodiments, the triterpenoic acids may comprise from about 1%to about 80% of the total active ingredients of the composition. In someembodiments, the triterpenoic acids may comprise from about 10% to about80% of the total active ingredients of the composition. In someembodiments, the triterpenoic acids may comprise from about 20% to about80% of the total active ingredients of the composition. In someembodiments, the triterpenoic acids may comprise from about 30% to about70% of the total active ingredients of the composition. In someembodiments, the triterpenoic acids may comprise from about 35% to about65% of the total active ingredients of the composition. In someembodiments, the triterpenoic acids may comprise from about 40% to about60% of the total active ingredients of the composition.

In some embodiments, the triterpenoic acids may comprise from about0.01% to about 80% of the total composition. In some embodiments, thetriterpenoic acids may comprise from about 0.01% to about 50% of thetotal composition. In some embodiments, the triterpenoic acids maycomprise from about 0.01% to about 10% of the total composition. In someembodiments, the triterpenoic acids may comprise from about 0.1% toabout 10% of the total composition. In some embodiments, thetriterpenoic acids may comprise from about 0.5% to about 4% of the totalcomposition. In some embodiments, the triterpenoic acids may comprisefrom about 0.1% to about 0.5% of the total composition. In someembodiments, the triterpenoic acids may comprise from about 0.1% toabout 1.0% of the total composition. In some embodiments, thetriterpenoic acids may comprise from about 0.1% to about 2% of the totalcomposition. In some embodiments, the triterpenoic acids may comprisefrom about 1% to about 3.5% of the total composition. In someembodiments, the triterpenoic acids may comprise from about 1.5% toabout 3% of the total composition. In some embodiments, the triterpenoicacids may comprise from about 1.75% to about 2.75% of the totalcomposition. In some embodiments, the triterpenoic acids may comprisefrom about 2% to about 2.5% of the total composition.

In some embodiments, the neutral triterpenoids may comprise from about1% to about 80% of the total active ingredients of the composition. Insome embodiments, the neutral triterpenoids may comprise from about 10%to about 80% of the total active ingredients of the composition. In someembodiments, the neutral triterpenoids may comprise from about 20% toabout 80% of the total active ingredients of the composition. In someembodiments, the neutral triterpenoids may comprise from about 30% toabout 70% of the total active ingredients of the composition. In someembodiments, the neutral triterpenoids may comprise from about 35% toabout 65% of the total active ingredients of the composition. In someembodiments, the neutral triterpenoids may comprise from about 40% toabout 60% of the total active ingredients of the composition.

In some embodiments, the neutral triterpenoids may comprise from about0.01% to about 80% of the total composition. In some embodiments, theneutral triterpenoids may comprise from about 0.01% to about 50% of thetotal composition. In some embodiments, the neutral triterpenoids maycomprise from about 0.01% to about 10% of the total composition. In someembodiments, the neutral triterpenoids may comprise from about 0.1% toabout 10% of the total composition. In some embodiments, the neutraltriterpenoids may comprise from about 0.5% to about 4% of the totalcomposition. In some embodiments, the neutral triterpenoid may comprisefrom about 0.1% to about 0.5% of the total composition. In someembodiments, the neutral triterpenoid may comprise from about 0.1% toabout 1.0% of the total composition. In some embodiments, the neutraltriterpenoid may comprise from about 0.1% to about 2% of the totalcomposition. In some embodiments, the neutral triterpenoids may comprisefrom about 1% to about 3.5% of the total composition. In someembodiments, the neutral triterpenoids may comprise from about 1.5% toabout 3% of the total composition. In some embodiments, the neutraltriterpenoids may comprise from about 1.75% to about 2.75% of the totalcomposition. In some embodiments, the neutral triterpenoids may comprisefrom about 2% to about 2.5% of the total composition.

In some embodiments, the composition for use in the invention comprisesa therapeutically effective amount of at least one triterpenoic acid andof at least one neutral triterpenoid as described herein, and apharmaceutically acceptable carrier. In some embodiments, the carrier ishydrophobic.

In some embodiments, the pharmaceutically acceptable carrier may includea hydrophobic carrier. In some embodiments, the hydrophobic carrier mayinclude at least one oil. In some embodiments, the oil may be selectedfrom the group consisting of a mineral oil, a vegetable oil andcombinations thereof. In some embodiments, the vegetable oil may beselected from the group consisting of cottonseed oil, olive oil, almondoil, canola oil, coconut oil, corn oil, grape seed oil, peanut oil,saffron oil, sesame oil, soybean oil, and combinations thereof. In someembodiments, the vegetable oil is a commercially available product whichmay be obtained either as a ‘NF’ (National Formulary) grade product oras a ‘USP’ (US Pharmacopoeia) grade product. In some embodiments, themineral oil may be light mineral oil. In some embodiments, thehydrophobic carrier may include at least one wax. In some embodiments,the hydrophobic carrier may include a combination of at least one oiland at least one wax.

The term “mineral oil” refers to a clear colorless nearly odorless andtasteless liquid obtained from the distillation of petroleum. It mayalso be referred to as white oil, white mineral oil, liquid petrolatum,liquid paraffin or white paraffin oil. In some embodiments, the mineraloil is light mineral oil, a commercially available product which may beobtained either as a ‘NF’ (National Formulary) grade product or as a‘USP’ (US Pharmacopoeia) grade product. For use in the invention, themineral oil is preferably free of aromatics and other unsaturatedcompounds.

The pharmaceutically acceptable carrier may alternately or in additioncomprise an oil replacement. Oil replacements include alkanes having atleast 10 carbon atoms (e.g., isohexadecane), benzoate esters, aliphaticesters, noncomodogenic esters, volatile silicone compounds (e.g.,cyclomethicone), and volatile silicone substitutes. Examples of benzoateesters include C₁₂-C₁₅ alkyl benzoate, isostearyl benzoate, 2-ethylhexyl benzoate, dipropylene glycol benzoate, octyldodecyl benzoate,stearyl benzoate, and behenyl benzoate. Examples of aliphatic estersinclude C₁₂-C₁₅ alkyl octonoate and dioctyl maleate. Examples ofnoncomodogenic esters include isononyl isononanoate, isodecylisononanoate, diisostearyl dimer dilinoleate, arachidyl propionate, andisotridecyl isononanoate.

Cyclomethicone is an evaporative silicone which may be included in thecarrier to assist in making the composition amenable to ejection from aspray dispenser.

The hydrophobic carrier may further comprise at least one wax. Waxesinclude for example, beeswax; vegetable waxes, sugar cane waxes, mineralwaxes, and synthetic waxes. Vegetable waxes include for example,carnauba, candelilla, ouricury and jojoba wax. Mineral waxes include forexample, paraffin wax, lignite wax, microcrystalline waxes andozokerites. Synthetic waxes include for example, polyethylene waxes.

Various formulations of the different combinations of triterpenoic acidsand neutral triterpenoids and preparation thereof are disclosed herein.The pharmaceutical compositions of the invention may be administered byany means that achieve their intended purpose. For example,administration may be by, for example, oral, parenteral, topical,transdermal routes, such as, for example, subcutaneous, intravenous,intramuscular, intradermal, intraperitoneal, intraarterial,intrauterine, intraurethral, intracardial, intracerebral,intracerebroventricular, intrarenal, intrahepatic, intratendon,intraosseus, intrathecal, dermal, vaginal, rectal, inhalation,intranasal, ocular, auricular and buccal administration routes.

The administering may in addition comprise a technique or means such aselectroporation, or sonication in order to assist in their delivery, forexample transdermally. Other techniques which may be employed includefor example, radio frequency or pressurized spray application.

The dosage administered may be dependent upon the age, health, andweight of the subject, the use of concurrent treatment, if any,frequency of treatment, and the nature of the effect desired. The amounttriterpenoids of the present invention in any unit dosage form comprisesa therapeutically effective amount which may vary depending on therecipient subject, route and frequency of administration.

In some embodiments, when MA is one of the ingredients in thecomposition, the amount of the MA of the total composition may be in therange of about 0% to about 15%. In some embodiments, the amount of theMA of the total composition may be in the range of about 0% to about7.5%. In some embodiments, the amount of the MA of the total compositionmay be in the range of about 0% to about 2.5%. In some embodiments, theamount of the MA of the total composition may be in the range of about0% to about 1%. In some embodiments, the amount of the MA of the totalcomposition may be in the range of about 0% to about 0.3%. In someembodiments, the amount of the MA of the total composition may about0.3%. In some embodiments, the amount of MA with respect to the totalamount of triterpenoids may be in the range of about 0% to about 50%. Insome embodiments, the amount of MA with respect to the total amount oftriterpenoids may be in the range of about 0% to about 25%. In someembodiments, the amount of MA with respect to the total amount oftriterpenoids may be in the range of about 0% to about 8%. In someembodiments, the amount of MA with respect to the total amount oftriterpenoids may be in the range of about 6% to about 8%.

In some embodiments, when OA is one of the ingredients in thecomposition the amount of the OA of the total composition may be in therange of about 0% to about 25%. In some embodiments, the amount of theOA of the total composition may be in the range of about 0% to about10%. In some embodiments, the amount of the OA of the total compositionmay be in the range of about 0% to about 5%. In some embodiments, theamount of the OA of the total composition may be in the range of about0% to about 1%. In some embodiments, the amount of the OA of the totalcomposition may be in the range of about 0% to about 0.5%. In someembodiments, the amount of the OA of the total composition may about0.5%. In some embodiments, the amount of OA with respect to the totalamount of triterpenoids may be in the range of about 0% to about 50%. Insome embodiments, the amount of OA with respect to the total amount oftriterpenoids may be in the range of about 0% to about 25%. In someembodiments, the amount of OA with respect to the total amount oftriterpenoids may be in the range of about 0% to about 11%. In someembodiments, the amount of OA with respect to the total amount oftriterpenoids may be in the range of about 9% to about 11%.

In some embodiments, when MDA is one of the ingredients in thecomposition the amount of the MDA of the total composition may be in therange of about 0% to about 25%. In some embodiments, the amount of theMDA of the total composition may be in the range of about 0% to about10%. In some embodiments, the amount of the MDA of the total compositionmay be in the range of about 0% to about 5%. In some embodiments, theamount of the MDA of the total composition may be in the range of about0% to about 2.5%. In some embodiments, the amount of the MDA of thetotal composition may be in the range of about 0.5% to about 1%. In someembodiments, the amount of MDA with respect to the total amount oftriterpenoids may be in the range of about 0% to about 50%. In someembodiments, the amount of MDA with respect to the total amount oftriterpenoids may be in the range of about 5% to about 35%. In someembodiments, the amount of MDA with respect to the total amount oftriterpenoids may be in the range of about 10% to about 26%. In someembodiments, the amount of MDA with respect to the total amount oftriterpenoids may be in the range of about 20% to about 26%.

In some embodiments, when IMDA is one of the ingredients in thecomposition, the amount of the IMDA of the total composition may be inthe range of about 0% to about 25%. In some embodiments, the amount ofthe IMDA of the total composition may be in the range of about 0% toabout 10%. In some embodiments, the amount of the IMDA of the totalcomposition may be in the range of about 0% to about 5%. In someembodiments, the amount of the IMDA of the total composition may be inthe range of about 0% to about 2.5%. In some embodiments, the amount ofthe IMDA of the total composition may be in the range of about 0.6% toabout 1%. In some embodiments, the amount of IMDA with respect to thetotal amount of triterpenoids may be in the range of about 0% to about50%. In some embodiments, the amount of IMDA with respect to the totalamount of triterpenoids may be in the range of about 5% to about 35%. Insome embodiments, the amount of IMDA with respect to the total amount oftriterpenoids may be in the range of about 12% to about 26%. In someembodiments, the amount of IMDA with respect to the total amount oftriterpenoids may be in the range of about 20% to about 26%.

In some embodiments, when MLA is one of the ingredients in thecomposition, the amount of the MLA of the total composition may be inthe range of about 0% to about 10%. In some embodiments, the amount ofthe MLA of the total composition may be in the range of about 0% toabout 3%. In some embodiments, the amount of the MLA of the totalcomposition may be in the range of about 0% to about 1%. In someembodiments, the amount of the MLA of the total composition may be inthe range of about 0% to about 0.5%. In some embodiments, the amount ofthe MLA of the total composition may be in the range of about 0% toabout 0.2%. In some embodiments, the amount of the MLA of the totalcomposition may about 0.2%. In some embodiments, the amount of MLA withrespect to the total amount of triterpenoids may be in the range ofabout 0% to about 25%. In some embodiments, the amount of MLA withrespect to the total amount of triterpenoids may be in the range ofabout 0% to about 15%. In some embodiments, the amount of MLA withrespect to the total amount of triterpenoids may be in the range ofabout 0% to about 4%. In some embodiments, the amount of MLA withrespect to the total amount of triterpenoids may about 4%.

In some embodiments, when IMLA is one of the ingredients in thecomposition, the amount of the IMLA of the total composition may be inthe range of about 0% to about 10%. In some embodiments, the amount ofthe IMLA of the total composition may be in the range of about 0% toabout 3%. In some embodiments, the amount of the IMLA of the totalcomposition may be in the range of about 0% to about 1%. In someembodiments, the amount of the IMLA of the total composition may be inthe range of about 0% to about 0.5%. In some embodiments, the amount ofthe IMLA of the total composition may be in the range of about 0% toabout 0.2%. In some embodiments, the amount of the IMLA of the totalcomposition may about 0.2%. In some embodiments, the amount of IMLA withrespect to the total amount of triterpenoids may be in the range ofabout 0% to about 25%. In some embodiments, the amount of IMLA withrespect to the total amount of triterpenoids may be in the range ofabout 0% to about 15%. In some embodiments, the amount of IMLA withrespect to the total amount of triterpenoids may be in the range ofabout 0% to about 4%. In some embodiments, the amount of IMLA withrespect to the total amount of triterpenoids may about 4%.

In some embodiments, when 3-OAc-MLA is one of the ingredients in thecomposition, the amount of the 3-OAc-MLA of the total composition may bein the range of about 0% to about 15%. In some embodiments, the amountof the 3-OAc-MLA of the total composition may be in the range of about0% to about 5%. In some embodiments, the amount of the 3-OAc-MLA of thetotal composition may be in the range of about 0% to about 3%. In someembodiments, the amount of the 3-OAc-MLA of the total composition may bein the range of about 0% to about 1%. In some embodiments, the amount ofthe 3-OAc-MLA of the total composition may be in the range of about 0%to about 0.2%. In some embodiments, the amount of the 3-OAc-MLA of thetotal composition may about 0.2%. In some embodiments, the amount of the3-OAc-MLA with respect to the total amount of triterpenoids may be inthe range of about 0% to about 25%. In some embodiments, the amount ofthe 3-OAc-MLA with respect to the total amount of triterpenoids may bein the range of about 0% to about 15%. In some embodiments, the amountof the 3-OAc-MLA with respect to the total amount of triterpenoids maybe in the range of about 0% to about 10%. In some embodiments, theamount of the 3-OAc-MLA with respect to the total amount oftriterpenoids may be in the range of about 0% to about 5%. In someembodiments, the amount of the 3-OAc-MLA with respect to the totalamount of triterpenoids may about 3%.

In some embodiments, when 3-OAc-IMLA is one of the ingredients in thecomposition, the amount of the 3-OAc-IMLA of the total composition maybe in the range of about 0% to about 15%. In some embodiments, theamount of the 3-OAc-IMLA of the total composition may be in the range ofabout 0% to about 5%. In some embodiments, the amount of the 3-OAc-IMLAof the total composition may be in the range of about 0% to about 3%. Insome embodiments, the amount of the 3-OAc-IMLA of the total compositionmay be in the range of about 0% to about 1%. In some embodiments, theamount of the 3-OAc-IMLA of the total composition may be in the range ofabout 0% to about 0.2%. In some embodiments, the amount of the3-OAc-IMLA of the total composition may about 0.2%. In some embodiments,the amount of the 3-OAc-IMLA with respect to the total amount oftriterpenoids may be in the range of about 0% to about 25%. In someembodiments, the amount of the 3-OAc-IMLA with respect to the totalamount of triterpenoids may be in the range of about 0% to about 15%. Insome embodiments, the amount of the 3-OAc-IMLA with respect to the totalamount of triterpenoids may be in the range of about 0% to about 10%. Insome embodiments, the amount of the 3-OAc-IMLA with respect to the totalamount of triterpenoids may be in the range of about 0% to about 5%. Insome embodiments, the amount of the 3-OAc-IMLA with respect to the totalamount of triterpenoids may about 3%.

In some embodiments, when 3-OAc-epi-MLA is one of the ingredients in thecomposition, the amount of the 3-OAc-epi-MLA of the total compositionmay be in the range of about 0% to about 15%. In some embodiments, theamount of the 3-OAc-epi-MLA A of the total composition may be in therange of about 0% to about 5%. In some embodiments, the amount of the3-OAc-epi-MLA of the total composition may be in the range of about 0%to about 3%. In some embodiments, the amount of the 3-OAc-epi-MLA of thetotal composition may be in the range of about 0% to about 1%. In someembodiments, the amount of the 3-OAc-epi-MLA of the total compositionmay be in the range of about 0% to about 0.2%. In some embodiments, theamount of the 3-OAc-epi-MLA of the total composition may about 0.2%. Insome embodiments, the amount of the 3-OAc-epi-MLA with respect to thetotal amount of triterpenoids may be in the range of about 0% to about25%. In some embodiments, the amount of the 3-OAc-epi-MLA with respectto the total amount of triterpenoids may be in the range of about 0% toabout 15%. In some embodiments, the amount of the 3-OAc-epi-MLA withrespect to the total amount of triterpenoids may be in the range ofabout 0% to about 10%. In some embodiments, the amount of the3-OAc-epi-MLA with respect to the total amount of triterpenoids may bein the range of about 0% to about 5%. In some embodiments, the amount ofthe 3-OAc-epi-MLA with respect to the total amount of triterpenoids mayabout 3%.

In some embodiments, when 3-OAc-epi-IMLA is one of the ingredients inthe composition, the amount of the 3-OAc-epi-IMLA of the totalcomposition may be in the range of about 0% to about 15%. In someembodiments, the amount of the 3-OAc-epi-IMLA of the total compositionmay be in the range of about 0% to about 5%. In some embodiments, theamount of the 3-OAc-epi-IMLA of the total composition may be in therange of about 0% to about 3%. In some embodiments, the amount of the3-OAc-epi-IMLA of the total composition may be in the range of about 0%to about 1%. In some embodiments, the amount of the 3-OAc-epi-IMLA ofthe total composition may be in the range of about 0% to about 0.2%. Insome embodiments, the amount of the 3-OAc-epi-IMLA of the totalcomposition may about 0.2%. In some embodiments, the amount of the3-OAc-epi-IMLA with respect to the total amount of triterpenoids may bein the range of about 0% to about 25%. In some embodiments, the amountof the 3-OAc-epi-IMLA with respect to the total amount of triterpenoidsmay be in the range of about 0% to about 15%. In some embodiments, theamount of the 3-OAc-epi-IMLA with respect to the total amount oftriterpenoids may be in the range of about 0% to about 10%. In someembodiments, the amount of the 3-OAc-epi-IMLA with respect to the totalamount of triterpenoids may be in the range of about 0% to about 5%. Insome embodiments, the amount of the 3-OAc-epi-IMLA with respect to thetotal amount of triterpenoids may about 3%.

In some embodiments, the amount of the masticadienonic acid (MDA) may bein the range of about 0.05% to about 20%. In some embodiments, theamount of the isomasticadienonic acid (IMDA) may be in the range ofabout 0.05% to about 20%. In some embodiments, the amount of theoleanonic acid (OA) may be in the range of about 0.05% to about 20%. Insome embodiments, the amount of the masticadienonic acid (MDA) may be inthe range of about 0.1% to about 10%. In some embodiments, the amount ofthe isomasticadienonic acid (IMDA) may be in the range of about 0.1% toabout 10%. In some embodiments, the amount of the oleanonic acid (OA)may be in the range of about 0.1% to about 10%. In some embodiments, theamount of the masticadienonic acid (MDA) may be in the range of about0.5% to about 12%. In some embodiments, the amount of theisomasticadienonic acid (IMDA) may be in the range of about 0.5% toabout 12%. In some embodiments, the amount of the oleanonic acid (OA)may be in the range of about 0.5% to about 12%. In some embodiments, theamount of the masticadienonic acid (MDA) may be in the range of about0.5% to about 15%. In some embodiments, the amount of theisomasticadienonic acid (IMDA) may be in the range of about 0.5% toabout 15%. In some embodiments, the amount of the oleanonic acid (OA)may be in the range of about 0.5% to about 15%.

In some embodiments, when NF-1 is one of the ingredients in thecomposition, the amount of the NF-1 of the total composition may be inthe range of about 0% to about 25%. In some embodiments, the amount ofthe NF-1 of the total composition may be in the range of about 0% toabout 10%. In some embodiments, the amount of the NF-1 of the totalcomposition may be in the range of about 0% to about 5%. In someembodiments, the amount of the NF-1 of the total composition may be inthe range of about 0% to about 1%. In some embodiments, the amount ofthe NF-1 of the total composition may about 0.5%. In some embodiments,the amount of NF-1 with respect to the total amount of triterpenoids maybe in the range of about 0% to about 50%. In some embodiments, theamount of NF-1 with respect to the total amount of triterpenoids may bein the range of about 5% to about 25%. In some embodiments, the amountof NF-1 with respect to the total amount of triterpenoids may be in therange of about 9% to about 13%.

In some embodiments, when NF-2 is one of the ingredients in thecomposition, the amount of the NF-2 of the total composition may be inthe range of about 0% to about 25%. In some embodiments, the amount ofthe NF-2 of the total composition may be in the range of about 0% toabout 10%. In some embodiments, the amount of the NF-2 of the totalcomposition may be in the range of about 0% to about 5%. In someembodiments, the amount of the NF-2 of the total composition may be inthe range of about 0% to about 1%. In some embodiments, the amount ofthe NF-2 of the total composition may about 0.5%. In some embodiments,the amount of NF-2 with respect to the total amount of triterpenoids maybe in the range of about 0% to about 50%. In some embodiments, theamount of NF-2 with respect to the total amount of triterpenoids may bein the range of about 5% to about 25%. In some embodiments, the amountof NF-2 with respect to the total amount of triterpenoids may be in therange of about 9% to about 13%.

In some embodiments, when NF-3 is one of the ingredients in thecomposition, the amount of the NF-3 of the total composition may be inthe range of about 0% to about 25%. In some embodiments, the amount ofthe NF-3 of the total composition may be in the range of about 0% toabout 10%. In some embodiments, the amount of the NF-3 of the totalcomposition may be in the range of about 0% to about 5%. In someembodiments, the amount of the NF-3 of the total composition may be inthe range of about 0% to about 1%. In some embodiments, the amount ofthe NF-3 of the total composition may about 0.5%. In some embodiments,the amount of NF-3 with respect to the total amount of triterpenoids maybe in the range of about 0% to about 50%. In some embodiments, theamount of NF-3 with respect to the total amount of triterpenoids may bein the range of about 5% to about 25%. In some embodiments, the amountof the NF-3 with respect to the total amount of triterpenoids may be inthe range of about 9% to about 13%. In some embodiments, the amount ofNF-3 with respect to the total amount of triterpenoids may be in therange of about 10-12%.

In some embodiments, when NF-4 is one of the ingredients in thecomposition, the amount of the NF-4 of the total composition may be inthe range of about 0% to about 25%. In some embodiments, the amount ofthe NF-4 of the total composition may be in the range of about 0% toabout 10%. In some embodiments, the amount of the NF-4 of the totalcomposition may be in the range of about 0% to about 5%. In someembodiments, the amount of the NF-4 of the total composition may be inthe range of about 0% to about 1%. In some embodiments, the amount ofthe NF-4 of the total composition may about 0.33%. In some embodiments,the amount of NF-4 with respect to the total amount of triterpenoids maybe in the range of about 0% to about 50%. In some embodiments, theamount of NF-4 with respect to the total amount of triterpenoids may bein the range of about 2.5% to about 25%. In some embodiments, the amountof NF-4 with respect to the total amount of triterpenoids may be in therange of about 6% to about 9%.

In some embodiments, when NF-P is one of the ingredients in thecomposition, the amount of the NF-P of the total composition may be inthe range of about 0% to about 15%. In some embodiments, the amount ofthe NF-P of the total composition may be in the range of about 0% toabout 7.5%. In some embodiments, the amount of the NF-P of the totalcomposition may be in the range of about 0% to about 2.5%. In someembodiments, the amount of the NF-P of the total composition may be inthe range of about 0% to about 1%. In some embodiments, the amount ofthe NF-P of the total composition may be in the range of about 0% toabout 0.33%. In some embodiments, the amount of the NF-P of the totalcomposition may about 0.33%. In some embodiments, the amount of NF-Pwith respect to the total amount of triterpenoids may be in the range ofabout 0% to about 50%. In some embodiments, the amount of NF-P withrespect to the total amount of triterpenoids may be in the range ofabout 0% to about 25%. In some embodiments, the amount of NF-P withrespect to the total amount of triterpenoids may be in the range ofabout 0% to about 7%. In some embodiments, the amount of NF-P withrespect to the total amount of triterpenoids may be in the range ofabout 6% to about 7%.

In some embodiments, when NF-A is one of the ingredients in thecomposition, the amount of the NF-A of the total composition may be inthe range of about 0% to about 10%. In some embodiments, the amount ofthe NF-A of the total composition may be in the range of about 0% toabout 3%. In some embodiments, the amount of the NF-A of the totalcomposition may be in the range of about 0% to about 1%. In someembodiments, the amount of the NF-A of the total composition may be inthe range of about 0% to about 0.5%. In some embodiments, the amount ofthe NF-A of the total composition may be in the range of about 0% toabout 0.25%. In some embodiments, the amount of the NF-A of the totalcomposition may about 0.25%. In some embodiments, the amount of NF-Awith respect to the total amount of triterpenoids may be in the range ofabout 0% to about 25%. In some embodiments, the amount of NF-A withrespect to the total amount of triterpenoids may be in the range ofabout 0% to about 15%. In some embodiments, the amount of NF-A withrespect to the total amount of triterpenoids may be in the range ofabout 0% to about 6%. In some embodiments, the amount of NF-A withrespect to the total amount of triterpenoids may be in the range ofabout 4% to about 6%.

In some embodiments, when NF-B is one of the ingredients in thecomposition, the amount of the NF-B of the total composition may be inthe range of about 0% to about 10%. In some embodiments, the amount ofthe NF-B of the total composition may be in the range of about 0% toabout 3%. In some embodiments, the amount of the NF-B of the totalcomposition may be in the range of about 0% to about 1%. In someembodiments, the amount of the NF-B of the total composition may be inthe range of about 0% to about 0.5%. In some embodiments, the amount ofthe NF-B of the total composition may be in the range of about 0% toabout 0.25%. In some embodiments, the amount of the NF-B of the totalcomposition may about 0.25%. In some embodiments, the amount of NF-Bwith respect to the total amount of triterpenoids may be in the range ofabout 0% to about 25%. In some embodiments, the amount of NF-B withrespect to the total amount of triterpenoids may be in the range ofabout 0% to about 15%. In some embodiments, the amount of NF-B withrespect to the total amount of triterpenoids may be in the range ofabout 0% to about 6%. In some embodiments, the amount of NF-B withrespect to the total amount of triterpenoids may be in the range ofabout 4% to about 6%.

In some embodiments, the pharmaceutically acceptable carrier may includea hydrophobic carrier. In some embodiments, the hydrophobic carrier mayinclude at least one oil. In some embodiments, the oil may be selectedfrom the group consisting of a mineral oil, a vegetable oil andcombinations thereof. In some embodiments, the vegetable oil may beselected from the group consisting of cottonseed oil, olive oil, almondoil, canola oil, coconut oil, corn oil, grape seed oil, peanut oil,saffron oil, sesame oil, soybean oil, and combinations thereof. In someembodiments, the mineral oil may be light mineral oil. In someembodiments, the hydrophobic carrier may include at least one wax. Insome embodiments, the hydrophobic carrier may include a combination ofat least one oil and at least one wax.

In some embodiments, the pharmaceutically acceptable carrier may be aphospholipid.

In some embodiments, the composition is a pharmaceutical composition. Insome embodiments, the composition may be in a form suitable foradministration by a route selected from the group consisting ofparenteral, transdermal, oral and topical.

In some embodiments, the composition may be in a form suitable fortopical administration. In some embodiments, the composition may be in aform suitable for oral administration. In some embodiments, thecomposition is in a form suitable for parenteral administration. In someembodiments, the composition may be in a form suitable foradministration by injection. In some embodiments, the composition is aparenteral formulation for administration by a route selected from thegroup consisting of subcutaneous, intravenous, intramuscular,intradermal, intraperitoneal, intraarterial, intracerebral,intracerebroventricular, intraosseus and intrathecal.

In some embodiments, the composition may be a parenteral formulation foradministration by subcutaneous route.

In some embodiments, the composition may be a parenteral formulation foradministration by intramuscular route.

In various embodiments, the composition may be formulated foradministration by a route selected from the group consisting of dermal,vaginal, rectal, inhalation, intranasal, ocular, auricular and buccal.

In some embodiments, the pharmaceutical composition may be in a formselected from the group consisting of a capsule, a tablet, a liposome, asuppository, a suspension, an ointment, a cream, a lotion, a solution,an emulsion, a film, a cement, a powder, a glue, an aerosol and a spray.In some embodiments, the capsule may be selected from the groupconsisting of a hard gelatin capsule and a soft gelatin capsule. In someembodiments, the emulsion is a nanoemulsion or a microemulsion.

In some embodiments, the formulation may include at least one of aninclusion complex, a nanoemulsion, a microemulsion, a powder, a lipidraft, a lipid microparticle, a dendrimer and a liposome. In someembodiments, the inclusion complex may include at least onecyclodextrin. In some embodiments, the at least one cyclodextrin mayinclude hydroxypropyl-β-cyclodextrin. In some embodiments, thenanoemulsion may include droplets having average particle size of lessthan 800 nm. In some embodiments, the droplets may include dropletshaving average particle size of less than 500 nm. In some embodiments,the droplets may include droplets having average particle size of lessthan 200 nm. In some embodiments, the powder may include a spray driedpowder. In some embodiments, the liposome may include a multilamellarvesicle. In some embodiments, the microemulsion may include a non-ionicsurfactant. In some embodiments, the non-ionic surfactant may beselected from the group consisting of a polyoxyl castor oil, apolyoxyethylene sorbitan fatty acid ester (polysorbates), a poloxamer, avitamin E derivative, a polyoxyethylene alkyl ether, a polyoxyethylenesterate, or saturated polyglycolyzed glyceride or combinations thereof.

In some embodiments, the composition may be disposed on the article ofmanufacture in the form of a coating. In some embodiments, the articleof manufacture may include a vessel, wherein the composition may bedisposed within the vessel. In some embodiments, the article ofmanufacture may be selected from the group consisting of a fabricarticle, a diaper, a wound dressing, a medical device, a needle orplurality of needles, a microneedle or plurality of microneedles, aninjection device and a spray dispenser. In some embodiments, the articleof manufacture may include a plurality of microneedles. In someembodiments, the medical device is selected from the group consisting ofa prosthetic, an artificial organ or component thereof, a valve, acatheter, a tube, a stent, an artificial membrane, a pacemaker, asensor, an endoscope, an imaging device, a pump, a wire and an implant.In some embodiments, the implant is selected from the group consistingof a cardiac implant, a cochlear implant, a corneal implant, a cranialimplant, a dental implant, a maxillofacial implant, an organ implant, anorthopedic implant, a vascular implant, an intraarticular implant and abreast implant.

In some embodiments, the composition may be suitable for administrationby a means selected from the group consisting of electroporation,sonication, radio frequency, pressurized spray and combinations thereof.

The pharmaceutical compositions of the invention may be manufactured ina manner which is itself known to one skilled in the art, for example,by means of conventional mixing, granulating, dragee-making, softgelencapsulation, dissolving, extracting, or lyophilizing processes.Pharmaceutical compositions for oral use may be obtained by combiningthe active compounds with solid and semi-solid excipients and suitablepreservatives, and/or antioxidants. Optionally, the resulting mixturemay be ground and processed. The resulting mixture of granules may beused, after adding suitable auxiliaries, if necessary, to obtaintablets, softgels, capsules, or dragee cores.

Suitable excipients are, in particular, fillers such as saccharides,e.g., lactose or sucrose, mannitol or sorbitol; cellulose preparationsand/or calcium phosphates, e.g., tricalcium phosphate or calciumhydrogen phosphate; as well as binders, such as starch paste, using,e.g., maize starch, wheat starch, rice starch, potato starch, gelatin,tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodiumcarboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,disintegrating agents may be added such as the above-mentioned starchesand also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate. Auxiliariesare flow-regulating agents and lubricants, e.g., silica, talc, stearicacid or salts thereof, such as magnesium stearate or calcium stearate,and/or polyethylene glycol. Dragee cores are provided with suitablecoatings which, if desired, are resistant to gastric juices. For thispurpose, concentrated saccharide solutions may be used, which mayoptionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethyleneglycol and/or titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. In order to produce coatings resistant togastric juices, solutions of suitable cellulose preparations, such asacetylcellulose phthalate or hydroxypropymethyl-cellulose phthalate, areused. Dye stuffs or pigments may be added to the tablets or drageecoatings, e.g., for identification or in order to characterizecombinations of active compound doses.

Other pharmaceutical compositions for oral use include push-fit capsulesmade of gelatin, as well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol.

Formulations for parenteral administration include suspensions andmicroparticle dispersions of the active compounds as appropriate. Insome embodiments, oily injection suspensions may be administered.Suitable lipophilic solvents or vehicles include fatty oils, e.g.,sesame oil, or synthetic fatty acid esters, e.g., ethyl oleate,triglycerides, polyethylene glycol-400, cremophor, or cyclodextrins.Injection suspensions may contain substances which increase theviscosity of the suspension include, e.g., sodium carboxymethylcellulose, sorbitol, and/or dextran. Optionally, the suspension may alsocontain stabilizers.

Pharmaceutical compositions can also be prepared using liposomescomprising the active ingredient. As is known in the art, liposomes aregenerally derived from phospholipids or other lipid substances.Liposomes are formed by mono- or multi-lamellar hydrated liquid crystalswhich are dispersed in an aqueous medium. Any non-toxic, physiologicallyacceptable and metabolisable lipid capable of forming liposomes can beused. In general, the preferred lipids are phospholipids and thephosphatidyl cholines (lecithins), both natural and synthetic. Methodsto form liposomes are known in the art, as disclosed for example, inPrescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, NewYork, N.Y. (1976) and in U.S. Pat. No. 7,048,943.

Formulations for topical administration include ointments. Suitablecarriers include vegetable or mineral oils, white petrolatum, branchedchain fats or oils, animal fats and waxes. The preferred carriers arethose in which the active ingredient is soluble. Stabilizers, humectantsand antioxidants may also be included, as well as agents imparting coloror fragrance, if desired. Ointments may be formulated for example, bymixing a solution of the active ingredient in a vegetable oil such asalmond oil with warm soft paraffin, and allowing the mixture to cool.

The pharmaceutical composition may comprise an oil-in-water emulsion ormicroemulsion in order to facilitate its formulation for oral,parenteral or topical use Such emulsions/microemulsions generallyinclude lipids, surfactants, optionally humectants, and water. Suitablelipids include those generally know to be useful for creatingoil-in-water emulsions/microemulsions, for example fatty acid glycerideesters. Suitable surfactants include those generally known to be usefulfor creating oil-in-water emulsions/microemulsions wherein lipids areused as the oil component in the emulsion. Non-ionic surfactants may bepreferred, such as for example, ethoxylated castor oil, phospholipids,and block copolymers of ethylene oxide and propylene oxide. Suitablehumectants, if used, include for example propylene glycol orpolyethylene glycol.

The pharmaceutical composition may be formulated in the form of a gel,such as a hydrogel formed from a gel-forming polymer such ascarrageenan, xanthan gum, gum karaya, gum acacia, locust bean gum, guargum. A hydrogel may be combined with an oil-in-water emulsion comprisingthe active ingredient.

The pharmaceutical composition may be formulated in the form of a cementsuch as those comprising polymethylmethacrylate (PMMA) or calciumphosphate. In some embodiments, the pharmaceutical composition may beformulated in the form of a powder.

In some embodiments, the present invention provides therapeutic uses andmethods of treating Alzheimer's disease (AD) in a subject in needthereof, comprising administering to a subject a therapeuticallyeffective amount of a composition comprising a combination of at leastone triterpenoic acid and at least one neutral triterpenoid. In someembodiments, the present invention provides therapeutic uses and methodsof treating Parkinson's Diseases (PD) in a subject in need thereofcomprising administering to a subject a therapeutically effective amountof a composition comprising a combination of at least one triterpenoicacid and at least one neutral triterpenoid. In some embodiments, thepresent invention provides therapeutic uses and methods of treatingvascular dementia (VD) in a subject in need thereof, comprisingadministering to a subject a therapeutically effective amount of acomposition comprising a combination of at least one triterpenoic acidand at least one neutral triterpenoid. In some embodiments, the presentinvention provides therapeutic uses and methods of treatingParkinson-plus syndrome in a subject in need thereof, comprisingadministering to a subject a therapeutically effective amount of acomposition comprising a combination of at least one triterpenoic acidand at least one neutral triterpenoid. In some embodiments, the presentinvention provides therapeutic uses and methods of treatingParkinsonisms in a subject in need thereof, comprising administering toa subject a therapeutically effective amount of a composition comprisinga combination of at least one triterpenoic acid and at least one neutraltriterpenoid. In some embodiments, the present invention providestherapeutic uses and methods of treating Multi System Atrophy (MSA) in asubject in need thereof, comprising administering to a subject atherapeutically effective amount of a composition comprising acombination of at least one triterpenoic acid and at least one neutraltriterpenoid. In some embodiments, the present invention providestherapeutic uses and methods of treating Progressive Supranuclear Palsy(PSP) in a subject in need thereof, comprising administering to asubject a therapeutically effective amount of a composition comprising acombination of at least one triterpenoic acid and at least one neutraltriterpenoid. In some embodiments, the present invention providestherapeutic uses and methods of treating tauopathic diseases andconditions in a subject in need thereof, comprising administering to asubject a therapeutically effective amount of a composition comprising acombination of at least one triterpenoic acid and at least one neutraltriterpenoid. In some embodiments, the present invention providestherapeutic uses and methods of treating Primary Age-related Tauopathy(PART) in a subject in need thereof, comprising administering to asubject a therapeutically effective amount of a composition comprising acombination of at least one triterpenoic acid and at least one neutraltriterpenoid. In some embodiments, the present invention providestherapeutic uses and methods of treating Pick's disease in a subject inneed thereof, comprising administering to a subject a therapeuticallyeffective amount of a composition comprising a combination of at leastone triterpenoic acid and at least one neutral triterpenoid.

The step of administering the compositions may comprise any acceptableroute including oral, topical, parenteral, and transdermal, such as, forexample, parenteral administration includes intravenous, intramuscular,subcutaneous, intradermal, intraperitoneal, intraarterial, intrauterine,intraurethral, intracardial, intracerebral, intracerebroventricular,intrarenal, intrahepatic, intratendon, intraosseus, intrathecal, dermal,vaginal, rectal, inhalation, intranasal, ocular, auricular and buccalroutes of administration.

In some embodiments, the method may be carried out prior to or followingimplantation of a medical device into the subject. In some embodiments,the method may be carried out prior to or following implantation of amedical device into the subject in order to treat theimplications/consequences of the condition. Medical devices include, butare not limited to a prosthetic, an artificial organ or componentthereof, a valve, a catheter, a tube, a stent, an artificial membrane, apacemaker, a sensor, an endoscope, an imaging device, a pump, a wire andan implant. Implants include, but are not limited to a cardiac implant,a cochlear implant, a corneal implant, a cranial implant, a dentalimplant, a maxillofacial implant, an organ implant, an orthopedicimplant, a vascular implant, an intraarticular implant and a breastimplant.

In some embodiments, the medical device is an organ implant, which mayin certain cases comprise autologous cells of the subject.

In some embodiments, the step of contacting comprises a means selectedfrom the group consisting of electroporation, sonication, radiofrequency, pressurized spray and combinations thereof.

In some embodiments, the step of contacting comprises establishingcontact between interstitial fluid and the composition. Contact betweeninterstitial fluid and the composition may be accomplished by piercingand/or teasing the dermis with a needle, a microneedle, or an apparatuscomprising a plurality of needles or microneedles. Such needles ormicroneedles are preferably non-hollow and may be fashioned in aplurality for example, on a comb or brush-like apparatus.

The method of the invention is suitable for application in humans andnon-human mammals.

The method of the invention may encompass use of an article ofmanufacture which incorporates the composition comprising thecombinations described herein.

The following examples are presented in order to more fully illustratecertain embodiments of the invention. They should in no way, however, beconstrued as limiting the broad scope of the invention. One skilled inthe art can readily devise many variations and modifications of theprinciples disclosed herein without departing from the scope of theinvention.

EXAMPLES

Isolation of Triterpenoic Acids and Neutral Triterpenoids from MasticGum:

Many of the compositions disclosed in this application are prepared bymixing together individual triterpenoic acid(s) and neutraltriterpene(s). These individual triterpenoic acid(s) and neutraltriterpenoids can either be extracted from a natural source such asMastic gum, or can be the product of a chemical synthesis. The actualorigin of these individual compounds does not influence the propertiesof the prepared pharmaceutical compositions prepared using theseindividual compounds. It is therefore understood that the proceduresgiven below for the isolation and synthesis of several individualtriterpenoic acids and individual neutral triterpenes are only limitedpractical examples and that a person skilled in the art may usedifferent isolation procedures and synthesis procedures for obtainingthese individual compounds.

The current invention relates to the unexpected biological andpharmaceutical properties of the disclosed pharmaceutical compositionscomprising triterpenoic acid(s) and neutral triterpenoid(s). Thecombination of triterpenoic acid(s) and neutral triterpenoid(s) resultsin an overall pharmaceutical activity which cannot be obtained by usingonly the triterpenoic acids or only the neutral triterpenoids.

Example 1A—Preparation of Isolated Acidic Fraction of Mastic Gum

To a 50 gram amount of mastic gum, absolute ethanol (800 ML) was added,and the mixture was left to stand for 24 hours. The mixture was shakenfor 30 minutes at 150 rpm and left to stand for two hours. The obtainedethanol solution was decanted from insoluble material into a 3 L roundbottom flask. To the insoluble material 400 ML of fresh ethanol wasadded and the mixture was shaken again 30 minutes at 150 rpm and wasleft to stand for 30 minutes. The obtained ethanol solution was decantedand added to the first ethanol solution. This step was repeated oncemore using 200 ML absolute ethanol. This provided 1.4 L of ethanolsolution. The ethanol was evaporated using a rotary evaporator, andn-hexane (1.2 Liter) was added to the remaining material, and themixture was shaken at 150 rpm for 4 hours. It was then left to stand for4 hours and the hexane solution was decanted from insoluble materialinto a 3 L Erlenmeyer. To the remaining insoluble material, 800 ML freshhexane was added and the mixture was shaken for 6 hours at 150 rpm andleft to stand for 12 hours. The hexane solution was decanted into the 3L Erlenmeyer flask containing the first 1.2 L of hexane solution. Thehexane was evaporated in a clean 3 L roundbottom flask to give about 30grams of extract. (Yields range typically from 50-70% depending on theage and particle size of the used Mastic gum.)

The obtained extracted material was subsequently dissolved in diethylether (500 ML) and extracted with a 5% aqueous sodium carbonate solution(4×100 ML), the basic aqueous layer and an oily/emulsion layer werecarefully separated form the diethyl ether layer. The diethyl etherlayer was then additionally extracted with 0.4 N aqueous sodiumhydroxide (3×100 ML) and the basic aqueous layer and an oily/emulsionlayer were again carefully separated from the diethyl ether layer. (Thisremaining diethyl ether layer is called diethyl ether layer Nr.I, andwill be used herein below in Example 1B). The two basic aqueous extracts(including oily/emulsion layers) were separately acidified to pH 1-2 byslow addition of 10% aqueous hydrochloric acid and were subsequentlyextracted with fresh diethyl ether (3×200 ML). The thus obtained etheralfractions were combined and dried over anhydrous sodium sulfate. Afterfiltering off the sodium sulfate, the diethyl ether was removed using arotary evaporator. This procedure gave ca. 15 gram of isolated acidicfraction of mastic gum as a white solid, corresponding to about 50%yield based on the intermediate extract obtained after theethanol/hexane extraction. This particular isolated acidic fractionobtained from mastic gum as described hereinabove is termed “AcidicMixture 1”.

Based on the starting 50 grams of Mastic gum, the yield for this acidicfraction is about 30%. Typical yields of this particular acidic fractionfrom mastic gum range from about 25% to about 35%. Without wishing to bebound to any theory or mechanism, these variations in yield can occurdue to natural (e.g. seasonal) fluctuations in the composition of theMastic gum and may also be influenced by age and storage conditions ofthe Mastic gum.

Example 1B: Isolation of the Neutral Fraction of Mastic Gum

The diethyl ether layer Nr.I obtained in Example 1A was transferred to aclean separatory funnel and washed with water (200 ML) and brine (150ML). It was then dried over anhydrous sodium sulfate. The sodium sulfatewas removed by filtration and the diethyl ether was evaporated using arotary evaporator. This gave about 15 grams of isolated neutral fractionas a white to off-white sticky solid (which will become a very viscousliquid above 35-40° C.). This is about 50% yield based on the extractobtained after the ethanol/hexane extraction presented in Example 1A.This particular isolated neutral fraction obtained from mastic gum asdescribed here is termed “Neutral Mixture 1”. Based on the starting 50grams of Mastic gum, the yield for this neutral fraction (“NeutralMixture 1”) is about 30%. Typical yields of this neutral fraction frommastic gum range from about 25 to about 35%.

The mass-balance of this particular acid-base extraction described hereis typically over 90% and often more than 95% based on the intermediateextract obtained after the ethanol/hexane extraction procedure. Theratio of the thus isolated acidic fraction (“Acidic Mixture 1”) toisolated neutral fraction (“Neutral Mixture 1”) is usually approaching1:1 (and nearly always within the 0.8:1.2 to 1.2:0.8 range).

Isolation of individual triterpenoic acids and neutral triterpenoidsfrom isolated acidic fractions and isolated neutral fractions can beaccomplished using standard column chromatography and HPLC-methods asknown to a person skilled in the art.

It is to be understood, and it is clear to a person skilled in the art,that other extraction protocols can be used to obtain different isolatedacidic fractions and isolated neutral fractions from suitable plantmaterials that can subsequently be used for the isolation oftriterpenoic acids and/or neutral triterpenoids.

Example 2—Synthesis of a Triterpenoic Acid and Some NeutralTriterpenoids Synthesis A: Preparation of Oleanonic Acid

Oleanonic acid was obtained in three steps from oleanolic acid.

Oleanolic acid was first converted to the corresponding methyl ester bytreatment with methyl iodide and potassium carbonate indimethylformamide (DMF). Oxidation of oleanolic acid methyl ester tooleanonic acid methyl ester was performed using Dess-Martin periodanereagentin dichloromethane (DCM). Hydrolysis of oleanonic acid methylester with lithium hydroxide in aqueous THF gave upon acidification thedesired oleanonic acid.

Oleanonic acid methyl ester: Oleanolic acid (3.66 g, 1.0 eq) wasdissolved in DMF (20.0 vol.). K₂CO₃ (3.3 g, 3.0 eq) was added andmixture was stirred for 10 minutes, then methyl iodide (0.75 ml, 1.5 eq)was added. Reaction mixture was carried out at room temperatureovernight (full conversion on TLC). K₂CO₃ was filtered off from reactionmixture and reaction was poured into ice water. White solid was filteredoff, washed with water and dried under reduced pressure to give desiredproduct (3.62 g, 96.0%).Methyl ester hydrolysis: To oleanonic acid methyl ester (1.0 g) in dryDMF (30 ml) dry LiCl (200 mg) was added and the mixture was stirredunder nitrogen at 50° C. for 6 hrs. Upon cooling to room temperature thereaction mixture was quenched by addition of 5% Na2CO3 solution (20 ml)and stirred overnight. Then 3% aqueous HCl was added till pH=2 and themixture was extracted with diethyl ether (3×50 ml). The combined etherlayers were washed with 0.5% HCl, and dried over sodium sulfate.Evaporation of the diethyl ether gave oleanonic acid as a white solid(0.73 g).

Synthesis B: Preparation of NF-A (Betulone)

NF-A was synthesized from betulin-28-acetate in two steps.

First, the 3-hydroxyl-group was oxidized to the corresponding ketonewith PCC in dichloromethane. This was followed by the hydrolysis of theC-28 acetate group to give the desired NF-A (Betulone).

Oxidation step: 28-acetyl betuline (3.2 g, 1.0 eq) was dissolved in DCM(40.0 vol.). Mixture was cooled in ice bath, then PCC (2.13 g, 1.5 eq)was added. Reaction mixture was warmed to room temperature and stirredovernight. Mixture was concentrated on silica gel and purified via flashcolumn chromatography eluted with hexane:EtOAc (95:5->90:10->85:15) togive desired product as white powder (2.85-3.15 g, 80.0-99%).Acetate hydrolysis: Starting material (1.14 g, 1.0 eq) was dissolved inmixture of THF:H₂O (2:1, 40.0 vol.) then LiOH monohydrate (0.57 g, 10.0eq) was added. Reaction was carried out at room temperature for 3 days.THF was evaporated. Mixture was extracted with EtOAc (3×), organiclayers were combined, dried over MgSO4 and then concentrated underreduced pressure. Crude product was purified via column chromatographyeluted with hexane:EtOAc 9:1 to give white powder (80%). Recoveredstarting material was hydrolyzed once more time, total yield 1.04 g(91.0%).

Synthesis C: Preparation of NF-B (oleanonic alcohol;28-hydroxy-beta-amyrone)

NF-B was synthesized from oleanonic acid methyl ester in three steps.

First, the 3-oxo group of oleanonic acid methyl ester (see synthesis Aabove) was converted with ethylene glycol and catalytic p-TosOH(p-Toluenesulfonic acid) to the corresponding acetal using the standardDean-Stark set-up with toluene as the solvent. Next the methyl estergroup was reduced to the corresponding alcohol with lithium aluminiumhydride in THF. Hydrolysis of the acetal with diluted aqueous HCl inacetone gave the desired NF-B (oleanonic alcohol).

Acetal formation: Oleanonic acid methyl ester (1.4 g, 1.0 eq) wasdissolved in toluene (20.0 vol.) then TsOH (0.006 g, 0.01 eq) andethylene glycol (0.46 g, 2.5 eq) were added. Reaction was refluxed for 3hours under Dean-Stark condenser. TLC indicated full conversion ofstarting material. Reaction was cooled to RT and quenched with NaHCO₃sat. sol., then extraction to EtOAc was done (3×). Organic layers werewashed with water, dried over MgSO₄ and concentrated. The crude productwas obtained as a grey solid and was used in the next step without anyfurther purification (1.42 g).Ester reduction: LAH (0.86 g, 2.5 eq) was suspended in THF anh. (20.0vol.) and was cooled to 0° C. Starting material (3.85 g, 1.0 eq) wasdissolved in THF anh. (25.0 vol.) and was added to suspension dropwise.After addition mixture was warmed to room temperature and stirred for 2hours (full conversion on TLC). Reaction mixture was quenched by “1-2-3method”. The resulting slurry was filtered through Celite. The filtratewas concentrated and used in the next step without further purification(3.5 g).

“1-2-3 Method”:

-   -   1. Add H₂O to reaction mixture. The same quantity (mL) of water        as quantity (g) of LAH    -   2. Add 15% NaOH to mixture. Double quantity (mL) of 15% NaOH as        quantity (g) of LAH    -   3. Add H₂O to mixture. Triple quantity (mL) of water as        quantity (g) of LAH        Acetal hydrolysis: Starting material (3.5 g, 1.0 eq) was        suspended in mixture of acetone (12.0 vol.) and 1M HCl (10.0        vol.). Reaction mixture was refluxed for 3 hours (full        conversion on TLC). Reaction mixture was quenched with NaHCO₃        sat. solution to pH 8, extracted with EtOAc (3×), dried over        MgSO4 and concentrated. The crude product was purified via        column chromatography, eluted with hexane:EtOAc (98:2→95:5→93:7)        and triturated with MeOH (1 g crude product/5 mL MeOH). The        precipitate was filtered off and dried in vacuo to give desired        product as white solid (2.63 g).

Synthesis D and E: Preparation of Oleanolic Alcohol (aka erythrodiol;28-hydroxy-beta-amyrin) and NF-3 (Oleanonic Aldehyde)

It was found that oleanolic alcohol (aka erythrodiol) was most easilysynthesized by reduction of oleanolic acid methyl ester (see synthesisA) with lithium aluminium hydride in THF. (Attempts to prepare thiscompound by direct reduction of oleanolic acid gave very low yields evenafter prolonged reaction times and using large excess of lithiumaluminium hydride.)

Oleanonic aldehyde (NF-3) was subsequently synthesized from oleanolicalcohol by oxidation using the Dess-Martin periodane reagent.

28-hydroxy-beta-amyrin (Erythrodiol): LAH (1.2 g, 3.0 eq) was suspendedin anhydrous THF (10.0 vol.) and was cooled to 0° C. Starting material(5.0 g, 1.0 eq) was dissolved in anhydrous THF (15.0 vol.) and was addedto suspension dropwise. After addition, the mixture was allowed to reachroom temperature and was further stirred for 2 hours (full conversion onTLC). The reaction was quenched by the “1-2-3 method” (see synthesis C).The resulting slurry was filtered through Celite. Filtrate wasconcentrated and the crude product was used in the next step withoutfurther purification (4.60 g).Oleanonic aldehyde (NF-3): 28-hydroxy-beta-amyrin (2.8 g, 1.0 eq) wasdissolved in DCM (20.0 vol.) then was added DMP (5.36 g, 2.0 eq).Reaction was carried out for 1 hour. Crude product was concentrated onsilica gel and purified via column chromatography eluted with hexane,then hexane:EtOAc (99:1→9:1) to give oleanonic aldehyde as a white solid(0.60 g).

Synthesis F: Preparation of Masticadienonic Aldehyde

Masticadienonic aldehyde was prepared from masticadienonic acid in threesteps. The methyl ester of masticadienonic acid was prepared usingdiazomethane in diethyl ether or by using trimethylsilyldiazomethane indichloromethane (DCM)/methanol. Reduction of the methyl ester withlithium aluminium hydride gave masticadienediol. The diol was thenconverted into masticadienonic aldehyde by oxidation with Dess-MartinPeriodane reagent.

Masticadienonic acid methyl ester: MDA (1 g, 1.0 eq) was dissolved inmixture of DCM (10.0 ml) and MeOH (10.0 ml) and 2M solution ofTMS-diazomethane in DCM (4.4 ml, 4.0 eq) was added dropwise within 30minutes. Color of the solution turned to light yellow, reaction mixturewas stirred for 30 minutes. Reaction progress was monitored on TLC(hexane:EA 4:1, visualized in pAA stain solution).The reaction was quenched by addition of few drops of AcOH until theyellowish color disappeared. The mixture was concentrated, dissolved inEA and washed with sat. NaHCO₃ and sat. brine. Organic layer was driedand concentrated to give desired MDA-methyl ester (1.02 g). Product wasused in subsequent step without further purification.Masticadienediol: LAH (0.21 g, 10.0 eq) was suspended in THF anh. (20.0vol.) and was cooled to 0° C. MDA (0.25 g, 1.0 eq) was dissolved in THFanh. (25.0 vol.) and was added to suspension dropwise within 15 minutes.After addition mixture was warmed to room temperature and stirred for 2hours (full conversion on TLC). Reaction mixture was quenched by “1-2-3method”. Resulted slurry was filtrated through Celite. Filtrate wasconcentrated and was purified via column chromatography eluted withappropriate eluent mixture (DCM:MeOH) to give masticadienediol (110 mg).The same reaction on 2.5 g scale gave 1.7 gr product. A mixture ofisomers was obtained, with the 3-beta-isomer as the main product(alpha/beta ratio ca. 5:1). The isomers can be further separated bypreparative HPLC.Masticadienonic aldehyde: masticadienediol (0.45 g, 1.0 eq) wasdissolved in DCM (20.0 vol.) then was added DMP (0.95 g, 2.2 eq).Reaction was carried out for 2 hours. Crude product was concentrated onsilica gel and purified via column chromatography eluted with hexane,then hexane:EtOAc (99:1→9:1) to give desired product as white solid (0.3g).

Synthesis G: Preparation of Isomasticadienonic Aldehyde

Isomasticadienonic aldehyde was synthesized from isomasticadienonic acidusing the same sequence of reactions as used for masticadienonicaldehyde in Synthesis E described above.

Isomasticadienonic acid methyl ester: IMDA (1 g, 1.0 eq) was dissolvedin mixture of DCM (10.0 ml) and MeOH (10.0 ml) and 2M solution of TMSdiazomethane (4.4 ml, 4.0 eq) was added dropwise within 30 minutes.Color of the solution turned to light yellow, reaction mixture wasstirred for 30 minutes. Reaction progress was monitored on TLC(hexane:EA 4:1, visualized in pAA stain solution).The reaction was quenched by addition of few drops of AcOH until theyellowish color disappeared. The mixture was concentrated, dissolved inEA and washed with sat. NaHCO₃ and sat. brine. Organic layer was driedand concentrated to give desired IMDA methyl ester (1.02 g). Product wasused in subsequent step without further purification.Isomasticadienediol: LAH (0.21 g, 10.0 eq) was suspended in THF anh.(20.0 vol.) and was cooled to 0° C. MDA (0.25 g, 1.0 eq) was dissolvedin THF anh. (25.0 vol.) and was added to suspension dropwise within 15minutes. After addition mixture was warmed to room temperature andstirred for 2 hours (full conversion on TLC). Reaction mixture wasquenched by “1-2-3 method”. Resulted slurry was filtrated throughCelite. Filtrate was concentrated and was purified via columnchromatography eluted with appropriate eluent mixture (DCM:MeOH) to giveisomasticadienediol (0.16 g, 65.0%). The same reaction on 2.5 g scalegave 1.55 gr product (61%). A mixture of isomers was obtained, with the3-beta-isomer as the main product (alpha/beta ratio ca. 5:1). Theisomers can be further separated by preparative HPLC.Isomasticadienonic aldehyde: isomasticadienediol (0.45 g, 1.0 eq) wasdissolved in DCM (20.0 vol.) then was added DMP (0.95 g, 2.2 eq).Reaction was carried out for 2 hours. Crude product was concentrated onsilica gel and purified via column chromatography eluted with hexane,then hexane:EtOAc (99:1→9:1) to give desired product as white solid (0.4g).

Synthesis H: Preparation of NF-2((8R)-3-Oxo-8-hydroxypolypoda-13E,17E,21-triene)

NF-2 was prepared from NF-1 by oxidation of the secondary hydroxyl groupto the ketone using Dess-Martin periodane reagent.

NF-2 ((8R)-3-Oxo-8-hydroxypolypoda-13E,17E,21-triene): NF-1 (0.90 g, 1.0eq) was dissolved in DCM (20.0 vol.) then was added DMP (1.90 g, 2.2eq). Reaction was carried out for 2 hours. Crude product wasconcentrated on silica gel and purified via column chromatography elutedwith hexane, then hexane:EtOAc (9:1→3:1) to give desired product aswhite solid (0.72 g).Other suitable oxidation methods for this reaction are theSwern-oxidation, pyridinium chlorochromate in DCM and the Oppenaueroxidation.

Synthesis I: Preparation of Beta-Amyrin

Beta-amyrin was prepared in five steps from oleanolic acid.

First, the 3-hydroxyl group was protected as TES-ether usingTES-triflate (TES=triethylsilyl). This was followed by reduction of themethyl ester to the corresponding alcohol using lithium aluminiumhydride, giving the monoprotected diol. The free hydroxyl group wasoxidized to the aldehyde using PCC (pyridinium chlorochromate). Thealdehyde group was converted to beta-amyrin in a three-step one-potsequence. First step was formation of the tosylhydrazide. Upon changingthe solvent system, the hydrazide was reduced by refluxing withsodiumborohydride which simultaneously removed the TES-protecting groupresulting in direct formation of the desired beta-amyrin.

Oleanolic Acid Methyl Ester:

Oleanolic acid (5.0 g, 1.0 eq) was dissolved in DMF (20.0 vol.). K₂CO₃(4.54 g, 3.0 eq) was added and mixture was stirred for 5-10 min, thenCH₃I (2.0 eq) was added. Reaction mixture was carried out at roomtemperature overnight (full conversion on TLC). K₂CO₃ was filtered offfrom reaction mixture and reaction was poured into ice water. Whitesolid was filtered off, washed with water and dried under reducedpressure. Crude product was used in the next step without anypurification (5.1 g).

TES Protection of 3-Hydroxyl Group:

Oleanolic acid methyl ester (5.1 g, 1.0 eq) was dissolved in DCM (20.0vol.) containing TEA (9.9 ml, 6.6 eq). The mixture was stirred for 15minutes and TESOTf (8.0 ml, 3.3 eq) was added dropwise. The reactionmixture was stirred overnight at RT until completion. (TLC hexane:EA;4:1). The mixture was diluted with 1M HCl and extracted with DCM (2×).Combined organic layers were dried and concentrated. The crude mixturewas purified by column chromatography (hexane:EA 98:2) to give desiredproduct as white solid. (6.6 g).

Ester Reduction:

LAH (1.29 g, 2.5 eq) was suspended in THF anh. (20.0 vol.) and wascooled to 0° C. Starting material (6.61 g, 1.0 eq) was dissolved in THFanh. (25.0 vol.) and was added to suspension dropwise. After addition,mixture was warmed to room temperature and stirred for 2 hours (fullconversion on TLC). Reaction was quenched by “1-2-3 method” (seeSynthesis C). The resulting slurry was filtered through Celite. Thefiltrate was concentrated and used in the next step without furtherpurification (4.55 g).

Alcohol Oxidation:

Mono-protected diol (1.0 g, 1.0 eq) was dissolved in DCM (20.0 vol.) andcooled to 0° C. To that was added PCC (0.58 g, 1.5 eq) and mixture wasstirred for 2 h at RT. Reaction progress was monitored on TLC (hexane:EA9:1). Reaction was concentrated on SiO₂ and purified via columnchromatography eluted with hexane:EA to give pure product (0.76 g).One-Pot Conversion of TES-Aldehyde Intermediate into Beta-Amyrin(Hydrazide Formation; Reduction; TES Cleavage):Starting material (0.62 g, 1.0 eq) was suspended in EtOH (26.0 vol.),p-toluenesulfonyl hydrazide (0.25 g, 1.2 eq) was added and mixture wasrefluxed overnight. Reaction progress was monitored on TLC (hexane:EA7:3). EtOH was concentrated and residue was dissolved in THF (33.0 vol.)and water (5.0 vol.) and NaBH₄ (0.42 g, 10.0 eq). Reaction was continuedat RT overnight and then 2 hours at reflux. Reaction was cooled down andportioned between water and EA, layers were separated, water layer wasextracted 2× with EA. Combined organic layers were dried andconcentrated to give crude residue. Crude reaction mixture purified viacolumn chromatography eluted with hexane:EA to give beta-amyrin as awhite solid (100 mg).

Synthesis J: Preparation of Beta-Amyrone

Beta-amyrone was prepared from beta-amyrin by oxidation of the hydroxylgroup to the corresponding ketone using pyridinium chlorochromate (PCC).Other suitable methods are the Dess-Martin reagent or Swern oxidation.

Via PCC-oxidation: Beta-amyrin (100 mg, 1.0 eq) was dissolved in DCM(20.0 vol.) and cooled to 0° C. To that was added PCC (76 mg, 1.5 eq)and mixture was stirred for 1 h at RT. Reaction progress was monitoredon TLC (hexane:EA 6:1). Reaction was concentrated on SiO₂ and purifiedvia column chromatography eluted with hexane:EA (60:1→20:1) to give pureproduct (63 mg).Via Dess-Martin Reagent: Starting material (100 mg, 1.0 eq) wasdissolved in DCM (20.0 vol.) then was added DMP (0.95 g, 2.2 eq).Reaction was carried out for 2 hours. Crude product was concentrated onsilica gel and purified via column chromatography eluted with hexane:EA(60:1→20:1) to give pure product to give desired product as white solid(0.67 g).

Synthesis K: Preparation of 28-Oxo-Lupen-3-One

28-oxo-lupen-3-one was synthesized from NF-A (betulone, see SynthesisB), by oxidation of the 28-hydroxyl group to the corresponding aldehydewith Dess-Martin periodane.

Starting material (1.0 g, 1.0 eq) was dissolved in DCM (20.0 vol.) thenwas added DMP (2.20 g, 2.2 eq). Reaction was carried out for 2 hours.Crude product was concentrated on silica gel and purified via columnchromatography eluted with hexane, then hexane:EtOAc (99:1→9:1) to givedesired product as white solid (0.74 g).

Other suitable oxidation methods for this reaction where theSwern-oxidation, pyridinium chlorochromate in DCM.

Synthesis L: Preparation of Oleanolic Aldehyde

Oleanolic aldehyde was prepared in two steps from the mono-protecteddiol intermediate from beta-amyrin Synthesis I.

The free hydroxyl group was oxidized to the corresponding aldehyde usingPCC or Dess-Martin Periodane. This was followed by removal of theTES-group with TBAF in aqueous THF to give the desired oleanolicaldehyde.

Via PCC-oxidation: The mono-protected diol (1.0 g, 1.0 eq) was dissolvedin DCM (20.0 vol.) and cooled to 0° C. To that was added PCC (0.58 g,1.5 eq) and mixture was stirred for 2 h at RT. Reaction progress wasmonitored on TLC (hexane:EA 9:1). Reaction was concentrated on SiO₂ andpurified via column chromatography eluted with hexane:EA to give pureproduct (0.76 g).Via Dess-Martin oxidation: The mono-protected diol (1.0 g, 1.0 eq) wasdissolved in DCM (20.0 vol.) then was added DMP (2.20 g, 2.2 eq).Reaction was carried out for 2 hours. Reaction progress was monitored onTLC (hexane:EA 9:1). The reaction mixture was concentrated on SiO₂ andpurified via column chromatography eluted with hexane:EA to give pureproduct (0.69 g).Removal of TES-group: (150 mg, 1.0 eq) was dissolved in THF (15.0 vol.)and cooled to 0° C. To that was added TBAF (113 mg, 2.0 eq) and mixturewas stirred overnight at RT. Reaction progress was monitored on TLC(hexane:EA 4:1). Reaction was concentrated on SiO₂ and purified viacolumn chromatography eluted with hexane:EA (9:1→6:1) to give pureproduct (86 mg).

Some suitable references for synthesis of several triterpenoidsencountered in the current application are:

D. Barton et al. J. Chem. Soc. 1956, 4150,

V. Domingo et al. J. Org. Chem. 74, 6151, 2009.

V. Domingo et al. Org. Biomol. Chem. 11, 559, 2013.

J. Justicia et al. Eur. J. Org. Chem. 10, 1778, 2004.

Example 3—Preparation of Pharmaceutical Compositions and Formulations

The pharmaceutical compositions as shown in Table 1 were prepared bymixing and dissolving the required amounts of triterpenoic acid(s) andneutral triterpenoid(s) in a suitable solvent (e.g. diethylether)followed by addition of the required amount of pharmaceuticallyacceptable carrier. The mixture was then shaken or stirred until ahomogeneous clear solution was obtained, and the suitable solvent (e.g.diethyl ether) was removed using vacuum (e.g. a rotary evaporator). Thisgave the desired pharmaceutical composition.

Table 1A shows the pharmaceutical compositions that were prepared usingdiethyl ether as solvent, and pharmaceutical grade (NF-grade) cottonseedoil (stabilized with ca. 900 ppm BHT) as the pharmaceutically acceptablecarrier.

TABLE 1A Cpd/ Entry A B C D E F G H I J K 1 Acidic-1 Neutral-2 2Acidic-1 NF-1 NF-2 NF-3 NF-4 NF-A NF-B NF-P 3 Acidic-1 NF-1 NF-2 NF-3NF-4 NF-A NF-B 4 Acidic-1 NF-1 NF-2 NF-3 NF-4 5 Acidic-1 NF-1 NF-2 NF-36 Acidic-1 NF-1 NF-2 7 Acidic-1 NF-1 8 Acidic-1 NF-2 9 Acidic-1 NF-3 10Acidic-2 Neutral-1 11 Acidic-2 Neutral-2 12 Acidic-2 NF-1 NF-2 NF-3 NF-4NF-A NF-B NF-P 13 Acidic-2 NF-1 NF-2 NF-3 NF-4 NF-A NF-A 14 Acidic-2NF-1 NF-2 NF-3 NF-4 15 Acidic-2 NF-1 NF-2 NF-3 16 Acidic-2 NF-1 NF-2 17Acidic-2 NF-1 NF-3 18 Acidic-2 NF-1 NF-4 19 Acidic-2 NF-1 20 Acidic-2NF-2 21 Acidic-2 NF-3 22 Acidic-2 NF-4 23 Acidic-2 NF-A 24 Acidic-2 NF-B25 MDA IMDA NF-1 NF-2 NF-3 NF-4 NF-A NF-B 26 MDA IMDA NF-1 NF-2 NF-3NF-4 NF-A NF-B NF-P 27 MDA IMDA NF-1 NF-2 NF-3 NF-4 NF-A NF-B NF-P MLAIMLA 28 MDA IMDA NF-1 NF-2 NF-A NF-B NF-P 29 MDA IMDA NF-1 NF-2 NF-ANF-B 30 MDA IMDA NF-3 NF-4 NF-A NF-B 31 MDA IMDA NF-1 NF-2 NF-3 NF-4 32MDA IMDA NF-1 NF-2 NF-3 33 MDA IMDA NF-1 NF-2 NF-4 34 MDA IMDA NF-1 NF-235 MDA IMDA NF-1 36 MDA IMDA NF-2 37 MDA IMDA NF-3 38 MDA IMDA NF-4 39MDA IMDA OA MA NF-1 NF-2 40 MDA IMDA OA MA NF-1 41 MDA IMDA OA MA NF-242 MDA IMDA OA MA NF-3 NF-4 NF-A NF-B NF-P 43 MDA IMDA OA MA NF-3 NF-444 MDA IMDA OA NF-1 NF-2 45 MDA IMDA OA NF-1 46 MDA IMDA OA NF-2Additional formulations containing 3-OAc-MLA, 3-OAc-IMLA, 3-Ac-epi-MLAand 3-OAc-epi-IMLA which have been prepared as indicated above areindicated in Table 1B.

TABLE 1B Entry A B C D E F G H I 47 MDA IMDA 3-OAc-MLA 3-OAc-IMLA OANF-1 NF-2 NF-3 NF-4 48 MDA IMDA 3-OAc-MLA 3-OAc-IMLA OA NF-1 NF-2 NF-ANF-B 49 MDA IMDA 3-OAc-MLA 3-OAc-IMLA OA NF-1 NF-2 50 MDA IMDA 3-OAc-MLA3-OAc-IMLA NF-1 NF-2 51 MDA IMDA 3-OAc-MLA 3-OAc-IMLA NF-1 NF-2 NF-3NF-4 52 MDA IMDA 3-OAc-MLA 3-OAc-IMLA NF-1 NF-2 NF-A NF-B 53 MDA IMDA3-OAc-MLA 3-OAc-IMLA OA MA NF-1 NF-2 54 MDA IMDA 3-OAc-MLA 3-OAc-IMLANF-1 NF-4 55 MDA IMDA 3-OAc-MLA 3-OAc-IMLA NF-1 NF-3 56 MDA IMDA3-OAc-MLA 3-OAc-IMLA NF-2 NF-4 57 MDA IMDA 3-OAc-MLA 3-OAc-IMLA NF-2NF-3 58 MDA IMDA 3-OAc-MLA 3-OAc-IMLA NF-1 NF-2 NF-3 59 MDA IMDA3-OAc-MLA 3-OAc-IMLA NF-1 60 MDA IMDA 3-OAc-MLA 3-OAc-IMLA NF-2 61 MDAIMDA 3-OAc-epi-MLA 3-OAc-epi-IMLA OA NF-1 NF-2 NF-3 NF-4 62 MDA IMDA3-OAc-epi-MLA 3-OAc-epi-IMLA NF-1 NF-2 NF-3 NF-4 63 MDA IMDA3-OAc-epi-MLA 3-OAc-epi-IMLA OA NF-1 NF-2 64 MDA IMDA 3-OAc-epi-MLA3-OAc-epi-IMLA NF-1 NF-2 65 MDA IMDA 3-OAc-epi-MLA 3-OAc-epi-IMLA NF-1NF-2 NF-3 66 MDA IMDA 3-OAc-epi-MLA 3-OAc-epi-IMLA NF-1 NF-4 67 MDA IMDA3-OAc-epi-MLA 3-OAc-epi-IMLA NF-1 NF-3 68 MDA IMDA 3-OAc-epi-MLA3-OAc-epi-IMLA NF-2 NF-3 69 MDA IMDA 3-OAc-epi-MLA 3-OAc-epi-IMLA NF-2NF-4 70 MDA IMDA 3-OAc-epi-MLA 3-OAc-epi-IMLA NF-1 71 MDA IMDA3-OAc-epi-MLA 3-OAc-epi-IMLA NF-2

Example 4—Synergistic Effect of Combinations of Neutral Triterpenoidsand Triterpenoic Acids in Rat tMCAO Stroke Model

The Middle Cerebral Artery occlusion (MCAO) model is a reliable modelfor stroke in rats and mimics the human condition. Generally, focalischemia results in localized brain infarction and is induced by middlecerebral artery occlusion (MCAO) in rats. Occlusion of MCA leads todamage to the sensorimotor cortex due to neuronal loss, whereas thelevel of this damage can be assessed by histology evaluation of theinfarct size and various behavior tests.

Transient middle cerebral artery (tMCAO) occlusion was performedaccording to the method described by R. Schmid-Elsaesser et al. Stroke.1998; 29(10):2162-2170. For the experiments described herein, theanimals are anesthetized using ketamine/xylazine solution. The animal'sneck is then shaved and a midline incision is made in the skin of theneck, and the tissue underneath is bluntly dissected. The right CommonCarotid Artery (CCA) was exposed through a midline neck incision andcarefully dissected free from surrounding nerves and fascia—from itsbifurcation to the base of the skull. The occipital artery branches ofthe ECA (External Carotid Artery) were then isolated, and these brancheswere dissected and coagulated. The ECA was dissected further distallyand coagulated along with the terminal lingual and maxillary arterybranches, which was then divided. The ICA (Internal Carotid Artery) wasisolated and carefully separated from the adjacent vagus nerve, and thepterygopalatine artery was ligated close to its origin with a 5-0 nylonsuture. Next a 4-0 silk suture was tied loosely around the mobilized ECAstump, and a 4 cm length of 4-0 monofilament nylon suture (the tip ofthe suture was blunted by using a flame, and the suture was coated withpolylysine, prior to insertion) was inserted through the proximal ECAinto the ICA and thence into the circle of Willis, effectively occludingthe MCA. The surgical wound was closed and the animals were returned totheir cages to recover from anesthesia. Two hours after occlusion ratswere re-anesthetized, monofilament was withdrawn to allow reperfusion,surgical wound was closed and rats were returned to their cages.

Two hours post occlusion just before reperfusion animals were subjectedto neurological evaluation using the “Neuroscore for exclusion criteria”(Chen J. et al. Stroke. 2001; 32(4):1005-1011.). Only animals with anoverall score of >10 were included in the study.

Immediately post reperfusion, three hours after stroke induction, ratswere treated with compositions A, B, C, D, E and F via subcutaneousinjection. The experiment was carried out with a total of 68 rats asspecified hereinbelow.

Forelimb and Hind Limb Placing Tests

For the forelimb-placing test, the examiner holds the rat close to atabletop and scores the rat's ability to place the forelimb on thetabletop in response to whisker, visual, tactile, or proprioceptivestimulation. Similarly, for the hind limb placing test, the examinerassesses the rat's ability to place the hind limb on the tabletop inresponse to tactile and proprioceptive stimulation. Separate sub-scoresare obtained for each mode of sensory input and added to give totalscores (for the forelimb placing test: 0=normal, 12=maximally impaired;for the hind limb placing test: 0=normal; 6=maximally impaired). Scoresare given in half-point increments (see below). Typically, there is aslow and steady recovery of limb placing behavior during the first monthafter stroke.

-   -   Fore limb placing test (0-12):        -   Whisker placing (0-2);        -   Visual placing (forward (0-2), sideways (0-2))        -   Tactile placing (dorsal (0-2), lateral (0-2))        -   Proprioceptive placing (0-2).

Evaluation was carried out (a) prior to the operation, (b) on day 15after stroke induction and (c) on day 58 after stroke induction.Difference in forelimb placing tests between days 58 and 15 (p<0.01) arepresented in FIG. 1. The high difference values in groups A, B, C, E andF. indicated a significant healing and recovered post-strokeneurological function in the triterpenoid-treated groups compared to theplacebo group D.

Neurological Scoring (Neuroscore):

The Modified Neurological Rating Scale (mNRS), or Neuroscore, wascarried out (a) prior to the operation, (b) on day 15 after strokeinduction and (c) on day 58 after stroke Difference in neuroscoresbetween days 58 and 15 (p<0.01) are presented in FIG. 2. The highdifference values in groups A, B, C, E and F. indicated a significanthealing and recovered post-stroke neurological function in thetriterpenoid-treated groups compared to the placebo group D.

Compositions as Used in tMCAO Stroke Model“Acidic Mixture 1” means the isolated acidic fraction of Mastic gum asprepared according to Example 1A. “Acidic Mixture 1” contains as maincompounds the following:

-   -   MA: Moronic acid (12-15%)    -   OA: Oleanonic acid (18-20%)    -   MDA: 24-Z-Masticadienonic acid (20-22%)    -   IMDA: 24-Z-Isomasticadienonic acid (22-26%)    -   3-beta-OAc-24-Z-masticadienolic acid (4-7%)    -   3-beta-OAc-24-Z-isomasticadienolic acid (4-7%)

It further contains a number of other triterpenoic acids in smallamounts, typically less than 5%. Possible triterpenoic acids that it maycontain are:

-   -   MLA: 3-beta-masticadienolic acid    -   IMLA: 3-beta-isomasticadienolic acid    -   Dihydromasticadienonic acid    -   Dihydroisomasticadienonic acid

“Acidic Mixture 2” contains the following compounds in % (w/w):

MA: Moronic acid (15%)

-   -   OA: Oleanonic acid (15%)    -   MDA: 24-Z-Masticadienonic acid (25%)    -   IMDA: 24-Z-Isomasticadienonic acid (30%)    -   3-beta-OAc-24-Z-masticadienolic acid (8%)    -   3-beta-OAc-24-Z-isomasticadienolic acid (7%)

In Table 2, “Acidic Mixture 1 (2.5%)” means a 2.5% (w/w) formulation ofthe acidic fraction as isolated in Example 1A in cottonseed oil.Likewise, “Acidic Mixture 2 (2.5%)” means a 2.5% (w/w) formulation of“Acidic Mixture 2” as defined above,

“Neutral Mixture 1” is the neutral fraction as prepared according toExample 1A,B;

“Neutral Mixture 2” contains the following neutral triterpenoids:

-   NF-1: (8R)-3-beta, 8-dihydroxypolypoda-13E,17E,21-triene-   NF-2: (8R)-3-Oxo-8-hydroxypolypoda-13E, 17E,21-triene-   NF-3: Oleanonic aldehyde-   NF-4: Tirucallol-   NF-P: Dipterocarpol (20-hydroxydammar-24-en-3-one)-   NF-A: (Betulon), 28-hydroxylup-20(29)-en-3-one-   NF-B: Oleanonic alcohol; (28-hydroxy-beta-amyrone)-   3-beta-hydroxy-13-alpha-malabarica-14(26), 17E,21-triene-   20-hydroxy-lupan-3-one-   28-Nor-17-hydroxylupen-3-one-   28-oxo-lupen-3-one-   28-nor-beta-amyrone-   Isomasticadienonic aldehyde-   Isomasticadienediol-   Masticadienediol-   Oleanolic aldehyde (28-oxo-beta-amyrin),-   3-beta-20-dihydroxylupane-   Masticadienonic aldehyde-   3-oxo-malabarica-14(26), 17E,21-triene-   Beta-amyrone-   Beta-amyrin-   Germanicol,

The concentrations (in cottonseed oil) of each compound/fraction in thedifferent compositions as used for the tMCAO model are presented inTable 2. Table 3 presents the number of animals per group and dosing.

TABLE 2 Group Administered (Entry nr. Table 1A) fraction/compound(s)Concentrations (% w/w) A (4) Acidic Mixture 1 + Acidic Mixture 1: 2.5%NF-(1,2,3,4) NF-(1,2,3): 0.5% each NF-4: 0.33% B (27) MDA + MDA, IMDA:1% each IMDA + NF-(1,2,3): 0.5% each NF-(1,2,3,4, P,A,B) + NF-(4, P):0.33% each MLA + NF-(A,B): 0.25% each IMLA MLA, IMLA: 0.2% each C (31)MDA + MDA, IMDA: 1% each. IMDA + NF-(1,2,3): 0.5% each NF-(1,2,3,4)NF-4: 0.33% D (n.a.) Placebo Cottonseed oil (incl. BHT stabilizer) E (2)Acidic Mixture 1 + Acidic Mixture 1: 2.5%; NF-(1,2,3,4,P,A,B) NF(1,2,3):0.50% each. NF(4, P): 0.33% each. NF(A,B): 0.25% each F (26) MDA + MDA;IMDA 1% each IMDA + NF-(1,2,3): 0.5% each NF-(1,2,3,4, P,A,B) NF-(4, P):0.33% each NF-(A,B): 0.25% each

TABLE 3 Group number of Dose, Treatment (# in Table 1A) rats (n) (twiceweekly) duration (days) A (4) n = 13 50 μl/rat 58 B (27) n = 12 50μl/rat 58 C (31) n = 10 50 μl/rat 58 D (n.a.) n = 11 50 μl/rat 58 E (2)n = 13 50 μl/rat 58 F (26) n = 9  50 μl/rat 58

The rats of the different groups were injected twice a week subcutaneouswith 25 microliters of designated test item. First injection was giventhree hours after stroke induction. As used herein, the term‘Formulation X’ refers to the formulation administered to the ratsincluded in Group X, as described herein and in Table 2, wherein X is A,B, C, D, E or F.

Results and Conclusion

Rats included in groups A-F were treated with Compositions A, B, C, Eand F, which include a combination of at least one triterpenoic acid andat least one neutral triterpenoid, or with placebo Composition D for 58days via twice weekly subcutaneous injections starting immediately afterreperfusion. During the study the neurological and somatosensoryfunctions were monitored in a battery of behavioral tests.

Some spontaneous stroke recovery of neurological functions was observedduring the 58 days follow-up after stroke induction. All testedcompositions including combinations of at least one triterpenoic acidand at least one neutral triterpenoid, showed enhanced recovery ofneurological function compared to the placebo group (Group D). Whencomparing the effect of compositions including combinations of terpenoidcompounds, Compositions A and C displayed stronger effects comparing tocompositions B, E and F (FIG. 1).

Sensory motor functions were also improved following the treatment witheach one of the compositions comprising at least one triterpenoic acidand at least one neutral triterpenoid. Treatment with Composition Ashowed the largest difference compared to placebo (Group D) (FIG. 2). Anenhanced improvement of sensory motor function compared to the placebotreated Group D was observed also for Compositions B, C, E and F,whereas the improvement was more pronounced for Compositions F and C.Unexpectedly, the observed therapeutic effects due to administration ofthe different compositions comprising at least one triterpenoic acid andat least one neutral triterpenoid, increased over time and were morepronounced towards day 58 after the operation.

General rats' health was identical in all groups, as all of them gainedweight at the same rate with no significant differences between them.

In view of these findings it can be concluded that compositions ofvarious combinations of molecules as disclosed in the current inventionare effective for the treatment of stroke and have the potential torestore damaged motor function and to improve somatosensory deficits forsubjects, who suffered a stroke.

Example 5—Glutamate-Induced Neurotoxicity Model

Ischemic or hemorrhagic stroke, traumatic brain injury (TBI) and otherbrain injuries are among the most devastating events patients maysuffer. Despite having differing etiologies they appear to coalescearound the same complex pathophysiology including: immune suppression,free radical-mediated toxicity, brain/neuron damage, infection,cytokine-mediated cytotoxicity, inflammation and activation of glialcells. All of these result in cognitive and/or physical deficits.

Excito-neurotoxicity after different brain damages mainly results fromexcessive glutamate release with subsequent excessive influx of Ca2+,primarily mediated by N-methyl D-aspartate (NMDA) glutamate receptors.Glutamate release induces excitotoxicity and contributes to thepathophysiology of numerous neurological diseases including ischemia,inflammation, epilepsy, and neurodegenerative diseases.

Glutamate excitotoxicity is an important mechanism of neuronal death ina wide range of neurological disorders. A well-establish model was usedto implement glutamate-induced excitotoxicity in primary cultures ofcortical neurons in order to evaluate the potential neuroprotectiveeffect of the compositions of the current invention.

Experimental Procedure

Compositions used:Table 1A, Entry nr. 25, herein called “Combination A”.Table 1A, Entry nr. 31, herein called “Combination B:Exerpt from Table 1A:

Entry A B C D E F G H I 25 MDA IMDA NF-1 NF-2 NF-3 NF-4 NF-A NF-B 31 MDAIMDA NF-1 NF-2 NF-3 NF-4

Cell Culture

Cortices were harvested from E19 Sprague-Dawley rat embryos. Tissueswere enzymatically and mechanically dissociated to obtain a homogenouscell suspension, and cells were plated at 20000 per well of four 96 wellplates in 200 μL neuronal medium.

Glutamate Induced Neurotoxicity:

Six (6) days after plating (DIV6) of primary cortical neurons in 200 μLof growth medium, test compounds were applied on neurons, by replacinghalf of the growth medium with 90 μL of medium containing a cytolysisprobe which is a cell-impermeant and high affinity nuclei acid stainingdye which is non-fluorescent in the absence of nuclei acids and exhibitsa strong fluorescence upon binding to DNA. Then, 10 μL of 20×concentrated compounds in oil were added to the cells. The time lapseimage acquisition was initiated 20 hours after the first treatmentfollowing a computer network problem that did not allow initiation ofacquisitions from the time of the treatment. From that time to the endof the experiments, time lapse images (1 image every 4 hours) of theneuronal culture were taken in phase contrast and in fluorescence forcytolysis monitoring. This was performed in 4 different plates inparallel, each containing the test compounds and controls.

Nine (9) days after plating (DIV9), half of the growth medium wasrenewed with 90 μL of medium containing the cytolysis probe, and 10 μLof the same 20× concentrated compounds prepared at day 6 was added tothe cells. At DIV10, half of the pretreated plates (2 plates) weretreated with 100 μM glutamate/10 μM glycine to induce excitotoxicity, byaddition of 10 μL of 21× concentrated glutamate solution. One glutamatetreated plate and non-glutamate treated plate further receivedtreatments with test compounds at DIV 11 and at DIV 14 following thesame schedule (half medium renewal with addition of 10 μL concentratedcompounds on cells). Cells were followed up to DIV 15, 24 hours afterthe last treatment. At the end of the experiments, all the cultures werepermeabilized so the cytolysis probe labeled all the cells allowingtotal cell counting. Cytolysis over time was rationalized to total cellnumber to yield a percentage of cytolysed cells over time. All theexperiments were performed in triplicate in the same experimentalsession.

Treatment Protocol:

Test compounds were tested with 2 treatment schedules:

-   -   2 pre-treatments at −96 and −24 hours before glutamate        treatments    -   2 pre-treatments at −96 and −24 hours before glutamate        treatments of other plates but no glutamate treatment.

Assay Endpoints/Analysis

Percentage of cytolyzed neurons over time (kinetics) from DIV 6 to DIV15. As the monitoring period was very long, in some cases, the maximumcytolysis during this period exceeded the 100% that was measured at theend after cell permeabilization, because toxic treatments can inducedead cell detachments from the culture surface. In these cases, themaximum value that was reached during the monitoring (higher than theend 100% cytolysis value) was considered as 100%. In primary cultures,there is spontaneous neuronal loss over time. The first data point wasconsidered at 0% cytolysis so only cell death appearing during themonitoring period was considered for homogeneity.

For specific glutamate effects assessments, some representations use thedata point before glutamate application as the new 0% cytolysis tospecifically assess glutamate effects without interference of formercytolysis events. Fixed time point graphs were also extracted fromkinetic analyses, areas under curves (AUC) of the cytolysis kineticcurves were calculated from the 72 h time point to the end of thekinetics.

Results:

As shown in FIGS. 3A-3B, Combination A effectively decreased theexcitotoxic effect of glutamate in a dose-dependent manner, from 0.025to 0.5% (FIGS. 3A-B).

As shown in FIGS. 4A-B, Combination B also showed a dose-dependentdecrease in glutamate excitotoxic effects from 0.05 to 0.5%.

Conclusions:

Excitotoxic insults elicited by 100 μM glutamate could be partially andsignificantly reversed by administration of Combination A or CombinationB between concentrations of 0.025% (for combination A) and 0.05% (forcombination B) to concentration 0.5% for both combinations.

Example 6—Tau-Hyperphosphorylation Assay—In-Vitro Model for AlzheimersDisease

Dementia is characterized by progressive deterioration of cognitivefunctioning, which means the loss of the ability to think, remember, orreason, as well as behavioral abilities. Various disorders and factorscontribute to the development of dementia. Neurodegenerative disorderssuch as Alzheimer disease (AD), frontotemporal disorders, and Lewy bodydementia result in a progressive and irreversible loss of neurons andbrain functions. In some dementias, a protein called tau was shown to behyper-phosphorylated and aggregates inside nerve cells in the brain,causing cell death. Disorders that are associated with an accumulationof tau are called tauopathies and Okadaic acid (OKA) is generally usedto trigger tauopathies in animal models as it was shown to increase tauphosphorylation in cultured cells. Using OKA in combination with kinaseinhibitors, is an established in vitro pharmacological model to identifycompounds responsible for phosphorylating specific residues. In order todetermine the therapeutic effect of the various combinations andformulations, in tau-related dementias, such Alzheimer disease, a modelof Tau hyperphosphorylation induced by okadaic acid (OKA) on mature ratcortical neurons.

Experimental Procedure and Treatment Schedule

Compositions used:Table 1A, Entry nr. 25, herein called “Combination A”.Table 1A, Entry nr. 31, herein called “Combination B:Exerpt from Table 1A:

Entry A B C D E F G H I 25 MDA IMDA NF-1 NF-2 NF-3 NF-4 NF-A NF-B 31 MDAIMDA NF-1 NF-2 NF-3 NF-4

Cell Culture—

Cortices were harvested from E19 Sprague-Dawley rat embryos. Tissueswere enzymatically and mechanically dissociated to obtain a homogenouscell suspension, and cells were plated in a 96 well plate.

Experimental Procedure—

After 9 days in vitro, for the 24-hour treatment wells, 10 μL of 20×concentrated compounds or vehicle were added to neurons. LiCl at 3 mMwas added as a positive control. After 24 hours, 10 nM OA was added tothe wells as a 11× concentrated solution (20 μL on 200 μL of medium). Inthe wells that were not pre-treated for 24 hours, test compounds orvehicle were added just before OKA addition (10 μL from the prepared 20×concentrated dose-responses). After 3 hours of OKA treatment in the cellincubator, half of the medium was discarded and 100 μL of 2%paraformaldehyde added to the wells for fixation. Cells were thenprocessed for double immunofluorescent labeling of Neurons (MAP2labeling) and Phospho-Tau (Phospho-PHF-tau pSer202+Thr205 Antibody(AT8)). Nuclei were labeled using Hoechst 33342 10 μM in PBS.

Assay Endpoints/Analysis—

Percentage of neurons with high phospho-Tau intensity for eachexperimental condition. This percentage is calculated with a thresholdabove which significant Tau hyperphosphorylation is considered inneurons. This threshold is set up by comparing non-treated and okadaicacid treated conditions, between the negative and positive neuronalpopulations on a cell by cell scatterplot (cytometric analysis) inFluofarma's CytoSurfer software.

Results: As demonstrated in FIG. 5A, the tested formulation (CombinationA) is shown to dose dependently decrease Tau hyperphosphorylation withsignificant effects of the 0.25 and 0.5% concentrations applied as a 24hour pre-treatment before Okadaic Acid treatment. As demonstrated inFIG. 5B, the tested formulation (Combination B) dose dependentlydecreased Tau hyperphosphorylation with significant effects of the 0.25and 0.5% concentrations applied as a 24 hour pre-treatment beforeOkadaic Acid treatment.

Example 7—In-Vivo Models for Alzheimer's Disease

Dementia is characterized by progressive deterioration of cognitivefunctioning, which means the loss of the ability to think, remember, orreason, as well as behavioral abilities. Alzheimer's disease (AD) is themajor cause of dementia whereas, vascular dementia, dementia with Lewybodies, fronto-temporal dementia etc also lead to memory deficits. Areliable animal model of memory loss with certain characteristics havebeen established in multiple ways by exposing the animals to apredetermined brain injury or intracranial infusion of certainneurotoxins. An aged rat model for Alzheimer's with memory deficitsdemonstrate pathological features and complex behaviors common to AD,provides a natural model of aging and cognitive decline and is a usefulchoice for testing investigational therapies targeting mechanisms ofneuroprotection, learning, and memory

Many rodent behavioral tasks have been developed which are highlysensitive to the cognitive behavioral symptoms seen in AD, and thesetests are extensively used as clinical diagnostic tools.

Among the numerous tasks designed for assessing distinct memoryprocesses, the social recognition task in the rat offers the opportunityto evaluate a form of short-term working memory in the domain of socialcognition, and its modification by pharmacological agents orphysiopathological states, such as aging s. Social cognition in humansis obviously of great importance and its deficits, e.g., during agingand Alzheimer's dementia, often have dramatic consequences for thepatient and their environment. Impairments in executive function arealso a commonly observed cognitive impairments in neuropsychiatricdisorders such as Schizophrenia, AD and Parkinson's disease. Cognitivedecline and AD are closely related to aging within the human population.Aged rats and mice with memory deficits demonstrate pathologicalfeatures and complex behaviors common to AD, providing an insightfulalternative to traditional models. Aging is characterized by aprogressive decline of cognitive performance, which has been partiallyattributed to structural and functional alterations of hippocampus.Cognitive decline and AD are closely related to aging within the humanpopulation. An aged rat model for AD with memory deficits demonstratepathological features and complex behaviors common to AD, hence providesa natural model of aging and cognitive decline and are a useful choicefor testing investigational therapies targeting mechanisms ofneuroprotection, learning, and memory. In this study, the formulationswere tested for potential cognition enhancing activity using the SocialRecognition and Delayed Alternation Tests in the aged rat.

Experimental Procedure Delayed Alternation Test in the Aged Rat(Acquisition)—

The experimental protocol is similar to that described in Porsolt et al(Drug Dev. Res., 35, 214-229, 1995).

Feeding Schedule—

5 days before starting the Delayed Alternation experiment, animals weresubmitted to restricted access to food (15 g per day) in order tohabituate them to the food deprivation schedule used during theexperiment. This food deprivation schedule continued throughout theexperiment. Animals received the 15 g food ration in their home cagesafter the last animal is tested. Before the beginning of thelever-pressing, they were also given several 45 mg food pellets (thoseused in the Delayed Alternation) to habituate them to this novel food.

Delayed Alternation experiment—This experiment includes 2 consecutivephases:— Acquisition of lever-pressing (single lever); and Acquisitionof delayed alternation (two levers).

Acquisition of Lever-Pressing—

The aim of this phase is to train animals to lever-press in order toreceive a food pellet reward. The animals are submitted to about 10lever-pressing acquisition sessions over 2 weeks (5 days per week) inthe experimental chambers according to a fixed ratio (FR1) schedule ofreinforcement. Reinforcement consists of a food pellet (45 mg) deliveredafter each lever-press. Animals are first submitted to lever-pressingacquisition sessions where a response on either the right or the leftlever resulted in the delivery of a food pellet. The levers are insertedin the chamber at the beginning of the session and are withdrawn at theend of the session. Afterwards, they are then subjected to severalsessions in which the left or the right lever is pseudo-randomlypresented every 5 seconds. A response on the lever results in theretraction of the lever and in the delivery of a food pellet. If theanimals did not press the lever within 30 seconds, the lever wasretracted without reinforcement and followed 5 seconds later by a newlever presentation. The house light comes on at the beginning of thesession and is extinguished at the end of the session. Session terminateafter 30 minutes or after the animal makes 50 lever responses. At theend of this phase between 80 and 100% of the animals acquire thelever-press response (i.e. they have made at least 20 responses duringthe final session). Animals which fail to learn are discarded from theexperiment. If some animals are close to establishing steadylever-pressing behavior, they will be given extra training with the aimof having at least 12 animals per group.

Acquisition of Delayed Alternation (Drug Test)—

Subsequent to lever-pressing acquisition, animals were submitted to 10delayed alternation sessions over 2 consecutive weeks (5 days per week).Each session consists of 36 trials separated by 10 seconds. Each trialconsists of presenting the animal first with one lever (left or right).When the animal presses on the lever, the animal is given a food pellet,the lever is retracted and 2.5 seconds later both levers are presented.The animal has to learn to press on the lever opposite to thatpreviously presented to gain a food reward (non-matching to sample). Ifthe animal does not respond to a one- or two-lever presentation within20 seconds, the lever(s) will be withdrawn without food reinforcementand the next trial starts 10 seconds later. The house light comes on atthe beginning of the session and is extinguished at the end of thesession. Sessions terminate after the animal has completed 36 trials, orafter 30 minutes have elapsed.

Behavioral parameter analyzed was the choice reaction time. Choicereaction time is the reaction times to each two-lever presentation,expressed as the mean value per animal per session.

Statistical Analysis—

Data obtained during the delayed alternation acquisition will beanalyzed by comparing aged control with young controls using unpairedStudent's t tests at each session. Data obtained during the delayedalternation acquisition will be analyzed by comparing testsubstance-treated groups with aged controls using a two-way analysis ofvariance (with group and session as factor) with repeated measures ateach session. The analysis will be followed by a one-way analysis ofvariance at each session in case of significant group or interactioneffect and will be completed by Dunnett's t tests when group effect willbe significant.

Social Recognition Test in the Aged Rat

The method, which detects facilitating effects of drugs on age-relatedmemory deficits, follows that described by Lemaire et al(Psychopharmacology, 115, 435-440, 1994). The experiment was performedusing unfamiliar open testing cage (46.5×26.5×18.5 cm) containingsawdust, one testing cage per experimental adult rat. Adult rat wasallowed to habituate to the testing cage for at least 5 minutes. Then,an unfamiliar juvenile rat was placed in the testing cage with the adultrat for 5 min. Following this first contact (C1), the juvenile wasremoved and the adult rats was returned to its housing cage. 30 minutesafter the adult was brought back to the same testing cage and the samejuvenile (familiar) was then placed in the testing cage once again for a5 minutes test session. The time the rat spends investigating (sniffing,grooming, licking, closely following) the juvenile at each contact wasrecorded. A recognition index (=C2/C1) was also calculated. Under suchconditions, a mature adult rat normally recognizes the juvenile asfamiliar, as indicated by a reduction in the duration of socialinvestigatory behavior at C2. Aged rats normally show amnesia in thistask as indicated by the absence of a decrease in the duration of socialinvestigatory behavior at C2. Data was analyzed by comparing theduration of social investigation at the second contact with the firstcontact for each group using paired Student's t tests. Data was analyzedby comparing aged controls with adult controls using unpaired Student'st tests. Data was analyzed by comparing test substances-treated groupswith aged controls using one-way ANOVA followed by Dunnett's t tests incase of significant effects.

Drug Testing Procedure—

15 rats per group are included at the beginning of the experiments.Combination A, B and C were evaluated at 1 dose, administered s.c. twiceweekly for 10 weeks before the delayed alternation experiment and thenup to the end of the Social Recognition test (i.e. 13 weeks in total).During behavioural testing (social recognition, lever pressing stage anddelayed alternation acquisition), administration is given after testing.The experiment includes an aged control and an adult/young controlgroups, receiving administrations of vehicle.

Results Delayed Alternation Test:

Combination A, administered s.c. twice weekly for 10 weeks, generallydecreased choice reaction times significantly so at Session 6 (p<0.05),(FIG. 6). Combination B, administered s.c. twice weekly for 10 weeks,also showed a pronounced tendency to decreased choice reaction times,however without reaching statistical significance.

Social Recognition Experiment

Combination A administered s.c. twice weekly over 12 weeks,significantly decreased the duration of investigation of the familiarjuvenile at the second contact, as compared with the first contact(−39%, p<0.05). Combination B Showed a tendency to decrease the durationof investigation of the familiar juvenile at the second contact, ascompared with the first contact (−21%, p=0.0675), (FIG. 7). Bothcombination A and B show a general trend to decrease the recognitionindex was not significantly modified as compared with aged controls (A:0.64 versus 0.95, NS; B: 0.69 versus 0.95, NS), (FIG. 8).

Conclusions

The results show that repeated s.c. administration of Combinations A andB in aged rats have a beneficial effects on age-related processing speeddeficits in the Delayed Alternation Test. In addition, the resultssuggest the presence of beneficial effects for Combination A, andCombination B administered s.c. twice weekly for 12 weeks on age-relateddeficits in the Social Recognition Test in the rat. Hence, takentogether the results indicated the beneficial effects of the testedcompositions in the Alzheimers model.

Example 8—Effect of Tested Formulations on 6-OHDA Induced Toxicity onMesencephalic Primary Cultures and TH Positive Neurons

Parkinson's disease (PD) is a debilitating neurodegenerative disordercharacterized by the progressive loss of dopaminergic (DA) neurons inthe substantia nigra pars compacta (SNc), leading to a marked dopamine(DA) depletion in striatum, the primary projection region, as well asextrastriatal nuclei of the basal ganglia. As tyrosine hydroxylase (TH)catalyses the formation of L-dihydroxyphenylalanine (L-DOPA), therate-limiting step in the biosynthesis of DA, the disease can beconsidered as a TH-deficiency syndrome of the striatum. Problems relatedto PD usually build up when vesicular storage of DA is altered by thepresence of either α-synuclein protofibrils or oxidative stress.

Neurotoxin induced-PD models are widely used to understand themechanisms of neuronal degeneration in PD. 6-Hydroxydopamine (6-OHDA) isa selective catecholaminergic neurotoxin and is widely used both in vivoand in vitro studies to generate PD models.

The study was designed to examine the effects of the tested formulationsin two different models. In the first part of the study, exposure ofmesencephalic primary cultures with 6-OHDA was used as a model system toexamine the cell viability and neuronal TH expression. In the secondpart of the study, a rat model of Parkinson Disease was induced byunilateral intra-striatum injection of the neurotoxin 6-OHDA. Thisinjection produces DA neuron loss on the injected side while sparing thecontralateral DA neurons.

This model was used to evaluate the various formulations effect on motorfunction and TH expression in the rat brain.

Several behavioral tests have been developed or adapted from otherneurological impairment models to elucidate the limb sensorimotordeficits associated with unilateral 6-OHDA-induced striatal dopaminedepletion, and are correlated with the degree of dopaminergicdegeneration. The affected limb functions can be compared with that ofthe intact limb, thereby increasing the sensitivity and repeatability ofassessment across days.

Experimental Procedure:

Compositions used:Table 1A, Entry nr. 25, herein called “Combination A”.Table 1A, Entry nr. 31, herein called “Combination B”.Table 1A, Entry nr. 34, herein called “Combination C”.Exerpt from Table 1A:

Entry A B C D E F G H I 25 MDA IMDA NF-1 NF-2 NF-3 NF-4 NF-A NF-B 31 MDAIMDA NF-1 NF-2 NF-3 NF-4 43 MDA IMDA NF-1 NF-2

Cell Culture—

Primary culture of rat mesencephalic neurons from E14 rat embryos,treated with 6-hydroxy dopamine. Mesencephalons were harvested from E14Sprague-Dawley rat embryos. Tissues were enzymatically and mechanicallydissociated to obtain a homogenous cell suspension, and cells wereplated at 100000 per well of a 96 well plate in 100 μL serum-containingneuronal medium.

6-OH DA Induced Neurotoxicity—

After mesencephalic neuron plating in 150 μL of medium, and after 3hours, neurons were treated with test compounds by addition of 10 μL of20× concentrated compounds in oil and with 10 μM 6-OH DA in growthmedium (5× concentrated in 40 μL of growth medium). After 72 hours, themedium was removed and medium containing a fluorescent cytolysis markerwas added to measure general cytolysis in the culture. Cells were thenfixed and immunologically labelled with a Tyrosine Hydroxylase (TH)specific antibody for dopaminergic neuron counting and neurite lengthassessment of dopaminergic neurons. Images were acquired with a 10×objective, stitching 9 images per well, using a Pathway 855 platform andanalyzed using the Neurite Outgrowth module of Metamorph software. Allthe experiments were performed in triplicate in the same experimentalsession.

Tested Formulations (Combinations)—

Test combinations dose responses were prepared in vehicle (Cottonseedoil) at 20 times (20×) the final tested concentrations by serialdilutions on the day of the first treatment. For treatments, 10 μL ofeach concentrated solution or vehicle was applied on 190 μL of neuronalgrowth medium. Test formulations were added just before 6-OH DAaddition, from freshly prepared dose-responses in cottonseed oil.

Final Concentrations: Combination A: 0.025%, 0.005%, 0.025%, 0.05%,0.25%, 0.5%, 1% Combination B: 0.025%, 0.005%, 0.025%, 0.05%, 0.25%,0.5%, 1% Combination C: 0.025%, 0.005%, 0.025%, 0.05%, 0.25%, 0.5%, 1%Endpoints:

Number of cytolysed cells per well at endpoint and number of TH positiveneurons. Results are shown as means+/−standard deviation (SD).Statistical analyses were performed using Student's t-tests forcomparing two means or One Way ANOVA followed by Dunnett's multiplecomparison test for comparing multiple means to the Vehicle treatedgroup.

Results

Combination A—

As shown in FIG. 9A—Combination A at 0.005, 0.025, 0.05 and 0.25%significantly decreased the number of cytolyzed cells following 72 hoursof 6-OH DA treatment.As shown in FIG. 9B, The number of TH positive neurons was increased at0.25, 0.5 and 1% compared to the 6-OH DA treated wells.

Combination B

As shown in FIG. 10A, Combination B at 0.0025, 0.005 and 0.025%significantly decreased the number of cytolyzed cells following 72 hoursof 6-OH DA treatment.As shown in FIG. 10B, the number of TH positive neurons was increased at0.005 and 1% compared to the 6-OH DA treated wells.

Combination C

As shown in FIG. 11A, Combination C at 0.025% significantly decreasedthe number of cytolyzed cells following 72 hours of 6-OH DA treatment.As shown in FIG. 11B, The number of TH positive neurons was increased at0.025, 0.05, 0.25, 0.5 and 1% compared to the 6-OH DA treated wells.

Conclusions

6-OHDA-induced neurodegeneration in vitro model was used to assess thepotential protective effects of Combination A, Combination B andCombination C. Combination A, Combination B and combination C were shownto reduce cell death in mesencephalic neurons and increased the numberof dopaminergic neurons after 72 hours of treatments, hence serve asprotectants.

Example 9—Therapeutic Effect of Tested Combinations in the Rat6-OHDA-Induced Parkinson Disease (PD) Model

Rat model of Parkinson Disease was induced by unilateral intra-striatuminjection of the neurotoxin 6-hydroxydopamine (6-OHDA). This injectionproduces dopaminergic (DA) neuron loss on the injected side whilesparing the contralateral DA neurons.

Test Procedures:

Compositions used:

Table 1A, Entry nr. 25, herein called “Combination A”.

Table 1A, Entry nr. 34, herein called “Combination C”.

Exerpt from Table 1A:

Entry A B C D E F G H I 25 MDA IMDA NF-1 NF-2 NF-3 NF-4 NF-A NF-B 34 MDAIMDA NF-1 NF-2

Disease Induction:

Animals are anesthetized using ketamine (10%; 0.1 ml/kg body weight) andxylazine (2%; 0.01 ml/kg). The animals are then stereotacticallyinjected into the right striatum with 2 μL of 6-OHDA at a concentrationof 20 mg/ml in 0.02% ascorbic acid. Lesion coordinations are setaccording to bregma and dura in mm: L-3.5 mm; AP-1 mm; DV-5.5 mm.Following the injection (injection rate: 2 μl/5 min) the injectingneedle is left for another 1 minute to avoid back flow and then slowlyretracted.

Paw Placement Test (Cylinder Test)—

This test assesses a rat's independent forelimb use to support the bodyagainst the walls of a cylindrical enclosure. The test takes advantageof the animals' innate drive to explore a novel environment by standingon the hindlimbs and leaning towards the enclosing walls. To performthis test, rats are placed individually in a glass cylinder (21 cmdiameter, 34 cm height) and wall exploration is recorded for 3 minutes.No habituation to the cylinder prior to recording is allowed. Wallexploration is expressed in terms of the ratio between the intact (R)and impaired legs (L) and calculated as the ratio of the intact leg(R)+1/2 of both forelimbs (1/2B) relative to the value of the intact leg(R)+the value of the impaired leg (L)+the value of both legs (B) wascalculated. The paw placement test was conducted on Day −1 to obtainbaseline data test was performed again 7 days after the injection of6-OHDA, and animals with a ratio≥0.6 were included in the study. Onstudy days 17, 24, 30, 43, 57 and 70 the animals were re-tested fortheir performance in the paw placement test.

Histological Analysis—Tyrosine Hydroxylase (TH)—

At the end of the study all animals are perfused with saline/heparin viathe left heart ventricle, and then are perfused with 4% formalin. Organsare collected into vials containing 4% formalin for 24-48 hours. Thetissues were then embedded in paraffin and 4 μm sections were cut fromthe Striatum region and Substantia Nigra (SNpc) region for IHC stainingusing anti-Tyrosine hydroxylase (TH) antibody and IHF staining usinganti-alpha-Synuclein and anti-Glial fibrillary acidic protein (GFAP).

Results:

Paw placement test—In each examination day the average difference inratio between that day to day 7 (day X-day 7) was calculated. Animalstreated with a Vehicle showed an increase in the difference throughoutthe study. The increase in the difference ratio was found to besignificantly higher from day 30 until the end of the study on day 70and it is and indicator for time-dependent progression of the disease.Treatment with combination A and C generally showed a lower average ofthe differences from day 7 at all time points, as compared to thevehicle group. The difference reached statistically significance at day43 and 57 for combination A. Differences for combination C weresignificantly lower from those of vehicle from day 24 to 70 (FIGS. 12Aand 12B, respectively). This implies that combination A and C are ableto significantly decrease disease progression.

Histological Analysis—Tyrosine Hydroxylase (TH):

Analysis of TH positive cells in SNpc of animals treated with 6-OHDAshowed statistically significant reduction in the number of TH-IR(immune-responsive) cells in the right hemisphere vs. the lefthemisphere, indicating of the pronounced effect of the 6-OHDA in the. Inaddition, statistically significant reduction in TH-IR cells was foundin the right hemisphere of the Vehicle group compared to the righthemisphere of the Naïve animals (FIG. 13A). Statistically significantincrease was found in the number of TH positive cells in the righthemisphere of combination C-treated group compared to the vehicle group(17.05±8.16 vs. 0.48±0.48, p<0.05; T-TEST). A trend of increase in TH-IRcell number in SNpc of the right hemisphere was found in animals treatedwith Combination A in comparison to the Vehicle Group (17.1±9.1 vs.0.48±0.48, for Group A vs. vehicle, respectively; p=0.055; T-test),(FIG. 13B).

Conclusions:

Treatment with combinations A and C starting 7 days after theadministration of 6-OHDA, improved the mobility of the impairedforelimb, measured by using the paw placement test. Interestingly, themean ratio of animals treated with combinations A and C was hardlychanged throughout the study, relatively to day 7, in comparison to thegradual changes in the vehicle group, which indicates diseaseprogression. Histological analysis of brains using anti-TH antibodyshowed the validity of the model as presented by reduction in the numberof TH-IR cells in the SNpc of the 6-OHDA injected right hemisphere vs.the intact left hemisphere. Moreover, the TH-positively stained Striatumof the left hemisphere in comparison to the mostly unstained Striatum ofthe Right hemisphere, also highlights the extent of damage of 6-OHDA inthe injection site. Combination C showed a statistically significantincrease in the TH staining in the right hemisphere, compared to thevehicle group. Combination A showed a clear trend of activity inincreasing the number of TH-IR cell in SNpc and the TH-positive area inthe striatum of the 6-OHDA injected right hemisphere.

Example 10—Vascular Dimentia—Effect of Various Compostions in Reversingthe Neurodegenerative Effects of Chronic Cerebral Hypoperfusion(Vascular Dementia) in a Rat Model

Cerebral lesions can be experimentally induced in rat brains bypermanent occlusion of both common carotid arteries which can affectcognitive function. This model is similar to vascular dementia and theexperimental technique decrease the blood flow in the cerebral cortexand hippocampus by up to 40-80% for several months, inducing certainlearning disorders. Thus this model is used to study the efficacy of thecompositions of the current invention in reversing the deficienciescaused by vascular dementia lesions.

A total of 40 animals are randomized into 4 groups: an untreated shamcontrol group, a vehicle control group and 2 groups with differentcompositions of the current invention (10-15 animals per group). Theyare randomized into 4 groups, an untreated sham control group, a vehiclecontrol and 2 treated groups. Ten microliters of each composition (incottonseed oil) or vehicle are administered subcutaneously twice weeklyat equivalent intervals, with the first dose administered 14 days afterinduction of vascular dementia.

The Morris water maze (MWM) test is sensitive to hippocampal function.The water maze task is performed to evaluate two CCA-related learningdeficits using the method described previously (Watanabe et al.,Cilostazol Stroke. 2006; 37(6):1539-1545). In a 160-cm diameter circularpool filled with 20-cm deep water, a circular transparent acrylicplatform is prepared, the top surface of which is 3 cm below the water.Rats are released facing the wall, and the time taken to escape to theplatform is recorded as the escape latency. Tests are performed on day 3before CCA occlusion and on days 14, 35, 56, 84 and 112 after CCAocclusion. On training days six training trials are conducted per daywith an inter-trial interval of 2 min. Animals are placed in the pool atone of six starting positions. In each training trial, the time and pathlength required to escape onto the hidden platform are recorded. Resultsof six training trials are averaged to obtain a single representativevalue, and the averages are used for final statistical analyses. Animalsthat found the platform are allowed to remain on the platform for 30sec. Animals that do not find the platform within 90 sec are softlyguided to the platform for 30 sec at the end of the trial.

Performance of the various animal groups, treated with the compositionsof the invention, vehicle treated animals and sham control animals aretested for frequency in platform location; the time spent in platformarea; the latency to find the platform; the frequency in zone 1location; the time spent in light part; the latency to find theplatform; and the velocity

Examples of Compositions Tested in the VD Model

“Acidic Mixture 1” means the isolated acidic fraction of Mastic gum asprepared according to Example 1A. “Acidic Mixture 1” contains as maincompounds the following:

-   -   MA: Moronic acid (12-15%)    -   OA: Oleanonic acid (18-20%)    -   MDA: 24-Z-Masticadienonic acid (20-22%)    -   IMDA: 24-Z-Isomasticadienonic acid (22-26%)    -   3-beta-OAc-24-Z-masticadienolic acid (4-7%)    -   3-beta-OAc-24-Z-isomasticadienolic acid (4-7%)

It further contains a number of other triterpenoic acids in smallamounts, typically less than 5%. Possible triterpenoic acids that it maycontain are:

-   -   MLA: 3-beta-masticadienolic acid    -   IMLA: 3-beta-isomasticadienolic acid    -   Dihydromasticadienonic acid    -   Dihydroisomasticadienonic acid

“Acidic Mixture 2” contains the following compounds in % (w/w):

-   -   MA: Moronic acid (15%)    -   OA: Oleanonic acid (15%)    -   MDA: 24-Z-Masticadienonic acid (25%)    -   IMDA: 24-Z-Isomasticadienonic acid (30%)    -   3-beta-OAc-24-Z-masticadienolic acid (8%)    -   3-beta-OAc-24-Z-isomasticadienolic acid (7%)    -   In Table 4, “Acidic Mixture 1 (2.5%)” means a 2.5% (w/w)        formulation of the acidic fraction as isolated in Example 1A in        cottonseed oil. Likewise, “Acidic Mixture 2 (2.5%)” means a 2.5%        (w/w) formulation of “Acidic Mixture 2” as defined above,

“Neutral Mixture 1” is the neutral fraction as prepared according toExample 1A,B;

“Neutral Mixture 2” contains the following neutral triterpenoids:

-   NF-1: (8R)-3-beta, 8-dihydroxypolypoda-13E,17E,21-triene-   NF-2: (8R)-3-Oxo-8-hydroxypolypoda-13E, 17E,21-triene-   NF-3: Oleanonic aldehyde-   NF-4: Tirucallol-   NF-P: Dipterocarpol (20-hydroxydammar-24-en-3-one)-   NF-A: (Betulon), 28-hydroxylup-20(29)-en-3-one-   NF-B: Oleanonic alcohol; (28-hydroxy-beta-amyrone)-   3-beta-hydroxy-13-alpha-malabarica-14(26), 17E,21-triene-   20-hydroxy-lupan-3-one-   28-Nor-17-hydroxylupen-3-one-   28-oxo-lupen-3-one-   28-nor-beta-amyrone-   Isomasticadienonic aldehyde-   Isomasticadienediol-   Masticadienediol-   Oleanolic aldehyde (28-oxo-beta-amyrin),-   3-beta-20-dihydroxylupane-   Masticadienonic aldehyde-   3-oxo-malabarica-14(26), 17E,21-triene-   Beta-amyrone-   Beta-amyrin-   Germanicol,

The concentrations (in cottonseed oil) of each compound/fraction in thedifferent compositions are presented in Table 4.

TABLE 4 Group Administered (nr. Entry in Table 1A) fraction/compound(s)Concentrations (% w/w) A (4) Acidic Mixture 1 + Acidic Mixture 1: 2.5%NF-(1,2,3,4) NF-(1,2,3): 0.5% each NF-4: 0.33% B (27) MDA + MDA, IMDA:1% each IMDA + NF-(1,2,3): 0.5% each NF-(1,2,3,4, P,A,B) + NF-(4, P):0.33% each MLA + NF-(A,B): 0.25% each IMLA MLA, IMLA: 0.2% each C (31)MDA + MDA, IMDA: 1% each. IMDA + NF-(1,2,3): 0.5% each NF-(1,2,3,4)NF-4: 0.33% D (n.a.) Placebo Cottonseed oil (incl. BHT stabilizer) E (2)Acidic Mixture 1 + Acidic Mixture 1: 2.5%; NF-(1,2,3,4,P,A,B) NF(1,2,3):0.50% each. NF(4, P): 0.33% each. NF(A,B): 0.25% each F (26) MDA + MDA;IMDA 1% each IMDA + NF-(1,2,3): 0.5% each NF-(1,2,3,4, P,A,B) NF-(4, P):0.33% each NF-(A,B): 0.25% each (25) MDA + MDA; IMDA 1% each IMDA +NF-(1,2,3): 0.5% each NF-(1,2,3,4,A,B) NF-4: 0.33%. NF-(A,B): 0.25% each(43) MDA + MDA; IMDA 1% each IMDA + NF-(1,2): 0.5% each NF-(1,2)

Example 11—Experimental Autoimmune Encephalomyelitis (EAE), a Model forHuman Multiple Sclerosis (MS)

Experimental autoimmune encephalomyelitis (EAE) is a good model forhuman multiple sclerosis (MS) research. EAE IS induced by immunizationof female Lewis rats with guinea pig MBP. This is an acute model of EAE.Lewis rats are immunized with myelin basic protein (MBP) in Freund'sadjuvant by subcutaneous injection in the sub-planter region of hindpaw. Clinical symptoms start developing around day 10 with loss of tailtone (“limp tail”) and hind leg paralysis by day 14. The peak period istypically around day 14/15, after which time the animals startrecovering.

Experimental Design:

Rats are treated with tested composition beginning on the day ofimmunization as “preventive treatment” to test the effect of testcomposition on the development of hind leg paralysis. Treatment may alsobe started at the onset of the first clinical sign of EAE to test theeffect of test article on the recovery from paralysis.

Rats are dosed twice a week via SC route of administration. Eight ratsare assigned to each of the groups (vehicle, treatment groups, as inTable 4, above) for a total of 48 animals. Animals are observed dailyfor body weight loss and clinical symptoms. Blood sample are taken forserum preparation and further investigation. The study takesapproximately 21 days to complete.

Reagents:

Mycobacterium tuberculosis H37Ra (MT), Difco, Code 231141Incomplete freund's adjuvant (IFA), Difco.Lyophilized guinea pig spinal cord homogenate (MBP), Sigma (M2295)

Preparation of Encephalitogenic Emulsion

IFA is enriched with MT up to 4 mg/ml.The MT powder will be crushed using pestle and mortar.Lyophilized MBP will be weighed and suspended in PBS to yield 0.5 mg/mLThe 0.5 mg/ml homogenate of MBP will be mixed with equal amount of CFA(4 mg/mL MT)

Induction of EAE:

IFA is enriched with MT up to 4 mg/ml. The MT powder is crushed usingpestle and mortar. Lyophilized MBP is weighed and suspended in PBS toyield 0.5 mg/mL. The 0.5 mg/ml homogenate of MBP is mixed with equalamount of CFA (4 mg/mL MT) and emulsified in two syringes connected withLeur lock.

Rats are anesthetized and emulsion is injected SC in the BT, each rat ata volume of 0.2 ml. Rats are scored for clinical signs of EAE and wereweighed each other day up to 21 d post immunization (p.i.) The signs arescored as described below.

The emulsion is injected SC in the BT, each rat at a volume of 0.2 mlTest Item ROA and doses:Rats are treated with the indicated composition beginning on the day ofimmunization as “preventive treatment” to test the effect of testedcomposition on the development of hind leg paralysis.

Evaluation of the EAE Clinical Signs (Table 5):

TABLE 5 Score Clinical Signs Description 0 Normal behavior No clinicalsigns 1 Tail weakness The tail is limp and droops. 2 Hind legs hypotoniaLimb pareses, wobbly walk- when the and weakness. rat walks the hindlegs are unsteady or it drags one hind leg. 3 Hind legs paralysis, Therat can't move its hind legs front legs normal and it drags them when itwalks. Forelegs are normal. 4 Hind legs paralysis, The rat can't moveits hind legs front legs weak and it drags them when it walks. Forelegsare weak. 5 Full paralysis The rat can't move at all. The rat isparalyzed. 6 Moribund/Death

Body Weights:

Body weights are recorded twice weekly throughout the entire study, onthe days of dosing.

Clinical Signs:

Clinical Signs are Recorded Twice Weekly Throughout the Entire Study.

Blood Samples:

At the end of the study final bleeding is performed, blood samples aretaken under full anesthesia from orbital sinus (as much as possible) andoptionally, also from the heart. The blood is kept at room temperaturefor at least an hour for clotting. After, the blood is centrifuged atroom temperature for 10 minutes at 1790 g (4000 RPM for centrifuge no.060). The serum (supernatant) is separated from the blood cells using asuitable pipette and transferred to eppendorf marked according to theStudy Protocol. Serum is stored in refrigerator (−70° C.) before sendingto sponsor.

Tissue Collection:

After sacrificing, animals are dissected. Brain and sciatic nerve arecollected from each animal and immediately placed into 4% PFA forfurther histopathological evaluation.

1. A method of treating a condition selected from Multi System Atrophy(MSA), Progressive Supranuclear Palsy (PSP), Parkinson-plus syndrome,Parkinsonisms, tauopathic diseases and conditions and Pick's disease,comprising administering to a subject in need thereof a pharmaceuticalcomposition comprising a combination of at least one triterpenoic acidand at least one neutral triterpenoid, and a pharmaceutically acceptablecarrier, wherein the triterpenoic acid is selected from masticadienonicacid (MDA), isomasticadienonic acid (IMDA) or both, wherein the neutraltriterpenoid is selected from(8R)-3-beta-8-dihydroxypolypoda-13E,17E,21-triene (NF-1),(8R)-3-Oxo-8-hydroxypolypoda-13E,17E,21-triene (NF-2) or both
 2. Themethod of claim 1, wherein the pharmaceutical composition furthercomprises at least one additional triterpenoic acid selected from thegroup consisting of masticadienolic acid (MLA), isomasticadienolic acid(IMLA), 3-O-acetyl masticadienolic acid, 3-O-acetyl epimasticadienolicacid, 3-O-acetyl isomasticadienolic acid, 3-O-acetylepi-isomasticadienolic acid, oleanonic acid (OA), moronic acid (MA) andcombinations thereof.
 3. The method of claim 1, wherein thepharmaceutical composition further comprises at least one additionalneutral triterpenoid selected from the group consisting of oleanonicaldehyde (NF-3), tirucallol (NF-4), 28-hydroxylup-20(29)-en-3-one(NF-A), 28-hydroxy-beta-amyrone (NF-B).
 4. The method of claim 3,wherein at least one of said neutral triterpenoids is selected from NF-3and NF-4.
 5. The method of claim 2, wherein the pharmaceuticalcomposition comprises at least two additional triterpenoic acids
 6. Themethod of claim 3, wherein the pharmaceutical composition comprises atleast two additional neutral triterpenoids.
 7. The method of claim 1,wherein the pharmaceutical composition is substantially devoid ofessential oils.
 8. The method of claim 1, wherein at least onetriterpenoic acid is obtained from a plant source.
 9. The method ofclaim 1, wherein at least one triterpenoic acid is obtained via chemicalsynthesis.
 10. The method of claim 1, wherein at least one neutraltriterpenoid is obtained via chemical synthesis.
 11. The method of claim1, wherein at least one neutral triterpenoid is obtained from a plantsource.
 12. The method of claim 8, wherein said plant source comprisesmastic gum.
 13. The method of claim 1, wherein said pharmaceuticallyacceptable carrier comprises at least one oil.
 14. The method of claim1, wherein the pharmaceutical composition is in a form suitable foradministration by a route selected from the group consisting ofparenteral, transdermal, oral and topical.
 15. The method according toclaim 3, wherein the pharmaceutical composition comprisespharmaceutically active ingredients consisting essentially of: MA, OA,MDA, IMDA, 3-O-acetyl masticadienolic acid, 3-O-acetylisomasticadienolic acid, NF-1, NF-2, NF-3, NF-4, NF-A and NF-B; and thepharmaceutically acceptable carrier.
 16. The method according to claim3, wherein the pharmaceutical composition comprises pharmaceuticallyactive ingredients consisting essentially of: MA, OA, MDA, IMDA,3-O-acetyl masticadienolic acid, 3-O-acetyl isomasticadienolic acid,NF-1, NF-2, NF-3 and NF-4; and the pharmaceutically acceptable carrier.17. The method according to claim 3, wherein the pharmaceuticalcomposition comprises pharmaceutically active ingredients consistingessentially of: OA, MDA, IMDA, 3-O-acetyl masticadienolic acid,3-O-acetyl isomasticadienolic acid, NF-1, NF-2, NF-3, NF-4, NF-A andNF-B; and the pharmaceutically acceptable carrier.
 18. The methodaccording to claim 3, wherein the pharmaceutical composition comprisespharmaceutically active ingredients consisting essentially of: OA, MDA,IMDA, 3-O-acetyl masticadienolic acid, 3-O-acetyl isomasticadienolicacid, NF-1, NF-2, NF-3 and NF-4; and the pharmaceutically acceptablecarrier.
 19. The method according to claim 4, wherein the pharmaceuticalcomposition comprises pharmaceutically active ingredients consistingessentially of: MDA, IMDA, MLA, IMLA, NF-1, NF-2, NF-3, NF-4, NF—P, NF-Aand NF-B; and the pharmaceutically acceptable carrier.
 20. The methodaccording to claim 3, wherein the pharmaceutical composition comprisespharmaceutically active ingredients consisting essentially of: MDA,IMDA, NF-1, NF-2, NF-3 and NF-4; and the pharmaceutically acceptablecarrier.
 21. The method according to claim 1, comprisingpharmaceutically active ingredients consisting essentially of: of MDA,IMDA, NF-1, NF-2; and the pharmaceutically acceptable carrier.